CN107922506A - Tissue factor approach restrainer antibody and application thereof - Google Patents

Tissue factor approach restrainer antibody and application thereof Download PDF

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Publication number
CN107922506A
CN107922506A CN201680048417.2A CN201680048417A CN107922506A CN 107922506 A CN107922506 A CN 107922506A CN 201680048417 A CN201680048417 A CN 201680048417A CN 107922506 A CN107922506 A CN 107922506A
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antibody
seq
antigen
cdr
binding fragment
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CN107922506B (en
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J·R·阿普加
S·贝纳德
G·J·卡尔旺
S·R·赫特
M·霍尔斯蒂
R·贾舒亚
M·吉恩
Z·S·尤奥
D·彼德曼
M·施塔尔
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Pfizer Inc
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Pfizer Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The present invention relates to specific binding TFPI and suppress its active antibody and its antigen-binding fragment.These antibody and fragment can be used for treatment bleeding disorder and shorten the clotting time.

Description

Tissue factor approach restrainer antibody and application thereof
Technical field
The present invention relates to the antibody of bind tissue factor approach restrainer (TFPI).
Background technology
Hemophilia A and B are respectively as caused by plasma protein factor VIII (FVIII) or factors IX (FIX) functional defect X-linkage genetic disease.Haemophiliachemophiliac clinical severity is related with the residual level of coagulation factor activity.<1% because Sub- activity is related with heavy phenotype, and osculant hemophilia is related with 2% to 5% factor active, it is light-duty and 5% to 40% because Sub- activity is related.
The nursing standard of these diseases is the clotting factor that missing is substituted by venoclysis.The replacement factor is typically Recombinant protein, such as Xyntha (Factor IX) or BeneFIX (FIX), but still use the plasma-derived products of various purity.With Substitute the treatment that the factor carries out and can be irregular (treating bleeding as needed when bleeding occurs), or preventative (pass through Factor level is maintained in protection domain to prevent bleeding).There is clear evidence to show, prophylactic treatment can prevent bleeding and blood The associated joint damage of friendly patient's principal pathogenetic.Effective prophylactic treatment needs to be injected intravenously the factor weekly 3 to 4 times, this Difficulty is caused to compliance and reduces quality of life.Due to manufacturing the complexity of clotting factor, treatment cost is also expensive.In addition, A large amount of patient (up to 32% be heavy hemophilia A patients) develops the neutralizing antibody for the factor applied, this by The patient for having mutation in these genes is considered as foreign protein.The therapeutic modality that these patients needs substitute, such as by-path factor, Factor VIIa (NovoSeven).
The alternative for the treatment of is by expanding complete extrinsic pathway come around the demand to substituting the factor.Hemophilia Patient stops the ability of bleeding with some by its complete extrinsic pathway；But this is not enough to stop massive haemorrhage (major bleed) or prevention hematostaxis.The extrinsic pathway is not enough to provide protection, because it is by tissue factor approach Inhibitor (TFPI) quick closedown.
Although WO 2010/017196 (Beyer Co., Ltd (Bayer)), WO 2011/109452 (Beyer Co., Ltd), WO 2014/ 144577 (Beyer Co., Ltd), WO 2010/072687 (Novo Nordisk Co., Ltd (Novo Nordisk)), 2012/001087 (promises of WO With Nuo De companies), WO 2014/140240 (Novo Nordisk Co., Ltd) and WO 2015/007880 (Novo Nordisk Co., Ltd) disclose With reference to the antibody of mankind TFPI, but they do not provide the antibody of the present invention, and the antibody has dives as new hemophilia In the feature of therapeutic agent.
Preventive protection can be provided to reduce clotting factor administration frequency at the same time, reduce factor usage amount, allow to substitute delivering Approach (such as subcutaneous) and the product with the relatively low risk for producing neutralizing antibody, will meet that haemophiliac is not expired largely The demand of foot.
The content of the invention
The present invention discloses and antibody (and its antigen binding fragment exemplified with bind tissue factor approach restrainer (TFPI) Section).
Those skilled in the art are used only normal experiment and will be recognized that or can determine present invention tool described in the invention Many equivalents of body embodiment.Such equivalent is expected to be included in following embodiment (E).
E1. a kind of separated antibody or its antigen-binding fragment, it specifically binds tissue factor approach restrainer (TFPI) epitope of Kunitz domains 2 (K2), wherein the epitope includes residue Ile105, Arg107 and Leu131 (root According to SEQ ID NO：2 numberings).
E2. the antibody of embodiment 1 or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment do not combine The Kunitz domains 1 (K1) of TFPI.
E3. embodiment 1 or 2 antibody or its antigen-binding fragment, wherein the epitope further includes one or more Selected from the residue of the following group：Cys106, Gly108, Cys130, Leu131 and Gly132 are (according to SEQ ID NO：2 numberings).
E4. the antibody or its antigen-binding fragment of any one of embodiment 1 to 3, wherein the epitope further include it is residual Base Cys106, Gly108, Cys130, Leu131 and Gly132 are (according to SEQ ID NO：2 numberings).
E5. the antibody or its antigen-binding fragment of any one of embodiment 1 to 4, wherein the epitope further includes one It is or multiple selected from the residue of the following group：Asp102, Arg112, Tyr127, Gly129, Met134 and Glu138 are (according to SEQ ID NO：2 numberings).
E6. the antibody or its antigen-binding fragment of any one of embodiment 1 to 5, wherein the epitope further includes Asp102, Arg112, Tyr127, Gly129, Met134 and Glu138 are (according to SEQ IDNO：2 numberings).
E7. the antibody or its antigen-binding fragment of any one of embodiment 1 to 6, wherein the epitope is not comprising one or more It is a to be selected from the residue of the following group：E100、E101、P103、Y109、T111、Y113、F114、N116、Q118、Q121、C122、 E123, R124, F125, K126 and L140 are (according to SEQ ID NO：2 numberings).
E8. the antibody or its antigen-binding fragment of any one of embodiment 1 to 7, wherein the epitope does not include：E100、 E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126 and L140 (according to SEQ ID NO：2 numberings).
E9. the antibody or its antigen-binding fragment of any one of embodiment 1 to 6, wherein the epitope is not comprising one or more It is a to be selected from the residue of the following group：D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126 and G128 are (according to SEQ ID NO：2 numberings).
E10. the antibody or its antigen-binding fragment of embodiment 1 to 6 and 9 any one, wherein the epitope does not include： D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126 and G128 are (according to SEQ ID NO：2 numberings).
E11. the antibody or its antigen-binding fragment of any one of embodiment 1 to 10, wherein the epitope is comprising one or more It is a to be selected from the residue of the following group：Asp102、Gly104、Ile105、Cys106、Arg107、Gly108、Arg112、Tyr127、 Gly129, Cys130, Leu131, Gly132, Asn133, Met134 and Glu138 are (according to SEQ ID NO：2 numberings), wherein institute Epitope residues are stated due to the interaction with the antibody or its antigen-binding fragment and are had in hiding surface region (BSA) There is non-zero change.
E12. the antibody of embodiment 11 or its antigen-binding fragment, wherein the epitope includes：Asp102、Gly104、 Ile105、Cys106、Arg107、Gly108、Arg112、Tyr127、Gly129、Cys130、Leu131、Gly132、Asn133、 Met134 and Glu138 are (according to SEQ ID NO：2 numberings).
E13. the antibody or its antigen-binding fragment of any one of embodiment 1 to 12, wherein the epitope is comprising one or more It is a to be selected from the residue of the following group：Asp102, Arg107, Arg112, Tyr127 and Leu131 are (according to SEQ ID NO：2 numberings), Wherein described epitope residues from the residue of the antibody or its antigen-binding fragment with participating in hydrogen bond.
E14. the antibody of embodiment 13 or its antigen-binding fragment, wherein the epitope includes：Asp102、Arg107、 Arg112, Tyr127 and Leu131 are (according to SEQ ID NO：2 numberings).
E15. the antibody or its antigen-binding fragment of any one of embodiment 1 to 14, wherein the epitope is comprising one or more It is a to be selected from the contact residues of the following group：Asp102、Gly104、Ile105、Cys106、Arg107、Gly108、Arg112、 Tyr127, Gly129, Cys130, Leu131, Gly132, Met134 and Glu138 are (according to SEQ ID NO：2 numberings).
E16. the antibody or its antigen-binding fragment of embodiment 15, wherein the epitope includes：Asp102、 Gly104、Ile105、Cys106、Arg107、Gly108、Arg112、Tyr127、Gly129、Cys130、Leu131、Gly132、 Met134 and Glu138 are (according to SEQ ID NO：2 numberings).
E17. the antibody or its antigen-binding fragment of any one of embodiment 1 to 16, it includes it is following due to TFPI phases Interaction and have in BSA non-zero change weight (H) chain and light (L) chain paratope (paratope) residue (according to Kabat is numbered)：H33Ala、H58Tyr、H95Leu、H96Gly、H97Ala、H98Thr、H99Ser、H100Leu、H100A Ser, L29Ala, L31Tyr, L91Tyr, L95A Ser and L95B Gly.
E18. the antibody or its antigen-binding fragment of any one of embodiment 1 to 17, it includes following contact residues (according to Kabat is numbered)：(1) H47 is Trp or Tyr；(b) H58 is Tyr；And (c) L91 is Tyr or Arg；And optionally include：(d) L96 is Gly or Asn.
E19. the antibody or its antigen-binding fragment of any one of embodiment 1 to 18, it includes following contact residues (according to Kabat is numbered)：(a) H33 is Ala, Asn, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val；(b) H47 is Trp or Tyr；(c) H50 is Ala, Arg, Gly, Lys, Met, Phe, Pro, Ser, Thr, Tyr or Val；(d) H51 be Ile, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；(e) H52 is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr Or Val；(f) H56 is Ser, Arg, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val； (g) H58 is Tyr；(h) H95 is Leu, Gln, Ile, Phe or Tyr；(i) H96 be Gly, Ala, Arg, Asn, Asp, Gln, Ile, Lys, Met, Phe, Pro, Ser, Thr or Val；(j) H97 be Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；(k) H98 be Thr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；(l) H99 is Ser, Ala, Gly, Phe or Pro；(m) H100 is Leu, Arg, His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr or Val；(n) H100A be Ser, Ala, Arg, Asn, Asp, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Ser, Thr or Trp；(o) L29 be Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr or Trp, Tyr, Val；(p) L31 be Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；(q) L91 is Tyr or Arg；(r)L95A Be Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；(s) L95B be Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；And (t) L95C be Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；And optionally include following residue：(u) L93 be Tyr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；And (v) L96 is Gly or Asn.
E20. the antibody or its antigen-binding fragment of any one of embodiment 1 to 18, it includes following contact residues (according to Kabat is numbered)：(a) H33 is Ala or Val；(b) H47 is Trp；(c) H50 is Ala；(d) H51 is Ile；(e) H52 be Ser, Arg, Lys, Phe or Tyr；(f) H56 is Ser, Arg or Lys；(g) H58 is Tyr；(h) H95 is Leu；(i) H96 be Gly, Ala, Arg, Asn, Lys, Pro, Ser or Val；(j) H97 is Ala；(k) H98 be Thr, His, Ile, Leu, Met, Phe or Tyr；(1) H99 is Ser；(m) H100 is Leu, Phe, Trp or Tyr；(n) H100A be Ser, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, Phe, Pro or Trp；(o) L29 is Ala；(p) L31 is Tyr；(q) L91 is Tyr；(r) L95A be Ser, Phe, Trp or Tyr；(s) L95B is Gly；And (t) L95C be Ser, Arg, Asn, Gln, Glu, Ile, Leu, Lys, Met, Phe, Trp, Tyr or Val；And optionally include following residue：(u) L93 is Ser；And (v) L96 is Gly.
E21. the antibody or its antigen-binding fragment of any one of embodiment 1 to 18, it includes following contact residues (according to Kabat is numbered)：(a) H33 is Ala, Val, His or Phe；(b) H47 is Trp or Tyr；(c) H50 be Ala, Thr, Ser or Phe；(d) H51 is Ile, Arg, Lys or Pro；(e) H52 is Ser, Phe, Arg or Tyr；(f) H56 be Ser, Lys, Tyr or Phe；(g) H58 is Tyr；(h) H95 is Leu, Ile, Gln or Phe；(i) H96 is Gly, Arg, Asn or Lys；(j) H97 is Ala, Leu, Tyr or Ile；(k) H98 is Thr, Tyr, Phe or His；(1) H99 is Ser, Pro, Ala or Phe；(m) H100 is Leu, Tyr, Trp or Phe；(n) H100A is Ser, Arg, Leu or Trp；(o) L29 is Ala, Glu, Asp or Gln；(p) L31 is Tyr, Glu, Asp or Trp；(q) L 91 is Ty or Arg；(r) L95A is Ser, Phe, Tyr or His；L95B is Gly, Glu, Asp Or Pro；And (t) L95C is Ser, Trp, Tyr or Phe；And optionally include following residue：(u) L93 be Ser, Glu, Asp or His；And (v) L96 is Gly or Asn.
E22. the antibody or its antigen-binding fragment of any one of embodiment 1 to 18, it includes following contact residues (according to Kabat is numbered)：H33Ala、H47Trp、H50Ala、H51Ile、H52Ser、H56Ser、H58Tyr、H95Leu、H96Gly、 H97Ala、H98Thr、H99Ser、H100Leu、H100A Ser、L29Ala、L31Tyr、L91Tyr、L95A Ser、L95B Gly And L95CSer；And optionally include following residue：L93Ser and L96Gly.
E23. the antibody or its antigen-binding fragment of any one of embodiment 1 to 22, it includes heavy chain variable region (VH), institute VH is stated to include：
(a) SEQ ID NO are included：The VH complementary determining regions 1 (CDR-H1) of 38 amino acid sequence；
(b) SEQ ID NO are included：The VH complementary determining regions 2 (CDR-H2) of 39 amino acid sequence；
And
(c) SEQ ID NO are included：The VH complementary determining regions 3 (CDR-H3) of 40 amino acid sequence.
E24. the antibody or its antigen-binding fragment of any one of embodiment 1 to 22, it includes SEQ ID NO：41 CDR-H1, CDR-H2 and CDR-H3 sequence.
E25. the antibody or its antigen-binding fragment of any one of embodiment 1 to 24, it includes mankind's VH3 frame sequences.
E26. the antibody or its antigen-binding fragment of any one of embodiment 1 to 24, it includes mankind's VH1 frame sequences.
E27. the antibody or its antigen-binding fragment of any one of embodiment 1 to 24, it includes mankind's VH5 frame sequences.
E28. the antibody or its antigen-binding fragment of any one of embodiment 1 to 24, it includes human reproduction system IGHV3- The VH frame sequences of 23 or IGHV1-69.
E29. the antibody or its antigen-binding fragment of any one of embodiment 1 to 24, it includes human reproduction system IGHV3-7 VH frame sequences.
E30. the antibody or its antigen-binding fragment of any one of embodiment 1 to 24, it includes mankind's VH system genitales to share Frame sequence.
E31. the antibody or its antigen-binding fragment of any one of embodiment 1 to 30, it includes VH, the VH contains and selects From with least 90% identical amino acid sequence of the amino acid sequence of the following group：SEQ ID NO：41st, 63 and 65.
E32. the antibody or its antigen-binding fragment of any one of embodiment 1 to 31, it includes VH, the VH, which contains, to be selected from With the amino acid sequence of the following group：SEQ ID NO：41st, 63 and 65.
E33. the antibody or its antigen-binding fragment of any one of embodiment 1 to 32, it includes VH, the VH contains SEQ ID NO：41 amino acid sequence.
E34. the antibody or its antigen-binding fragment of any one of embodiment 1 to 32, it includes VH, the VH contains SEQ ID NO：63 amino acid sequence.
E35. the antibody or its antigen-binding fragment of any one of embodiment 1 to 32, it includes VH, the VH contains SEQ ID NO：65 amino acid sequence.
E36. the antibody or its antigen-binding fragment of any one of embodiment 1 to 35, it includes light chain variable region (VL), institute VL is stated to include：
(a) SEQ ID NO are included：The VL complementary determining regions 1 (CDR-L1) of 33 amino acid sequence；
(b) SEQ ID NO are included：The VL complementary determining regions 2 (CDR-L2) of 34 amino acid sequence；
And
(c) SEQ ID NO are included：The VL complementary determining regions 3 (CDR-L3) of 35 amino acid sequence.
E37. the antibody or its antigen-binding fragment of any one of embodiment 1 to 35, it includes SEQ ID NO：36 CDR-L1, CDR-L2 and CDR-L3 sequence.
E38. the antibody or its antigen-binding fragment of any one of embodiment 1 to 37, it includes mankind VKFrame sequence.
E39. the antibody or its antigen-binding fragment of any one of embodiment 1 to 37, it includes mankind VλFrame sequence.
E40. the antibody or its antigen-binding fragment of any one of embodiment 1 to 37, it includes human reproduction system IGKV3- 20 VL frame sequences.
E41. the antibody or its antigen-binding fragment of any one of embodiment 1 to 37, it includes human reproduction system IGKV1- 39 VL frame sequences.
E42. the antibody or its antigen-binding fragment of any one of embodiment 1 to 37, it includes mankind's VL system genitales to share Frame sequence.
E43. the antibody or its antigen-binding fragment of any one of embodiment 1 to 42, it includes VL, the VL contain with SEQ ID NO：36 at least 90% identical amino acid sequences.
E44. the antibody or its antigen-binding fragment of any one of embodiment 1 to 43, it includes VL, the VL contains SEQ ID NO：36 amino acid sequence.
E45. the antibody or its antigen-binding fragment of any one of embodiment 1 to 44, it includes heavy chain constant region (CH), institute CH is stated to contain and SEQ ID NO：20 at least 90% identical amino acid sequences.
E46. the antibody or its antigen-binding fragment of any one of embodiment 1 to 45, it includes CH, the CH contains SEQ ID NO：20 amino acid sequence.
E47. the antibody or its antigen-binding fragment of any one of embodiment 1 to 46, it includes constant region of light chain (CL), institute CL is stated to contain and SEQ ID NO：26 at least 90% identical amino acid sequences.
E48. the antibody or its antigen-binding fragment of any one of embodiment 1 to 47, it includes CL, the CL contains SEQ ID NO：The CL of 26 amino acid sequence.
E49. the antibody or its antigen-binding fragment of any one of embodiment 1 to 48, it includes Fc domains.
E50. the antibody or its antigen-binding fragment of embodiment 49, wherein the Fc domains are the Fc structures of IgA Domain.
E51. the antibody or its antigen-binding fragment of embodiment 50, wherein the IgA is IgA1Or IgA2。
E52. the antibody or its antigen-binding fragment of embodiment 49, wherein the Fc domains are the Fc structures of IgD Domain.
E53. the antibody or its antigen-binding fragment of embodiment 49, wherein the Fc domains are the Fc structures of IgE Domain.
E54. the antibody or its antigen-binding fragment of embodiment 49, wherein the Fc domains are the Fc structures of IgM Domain.
E55. the antibody or its antigen-binding fragment of embodiment 49, wherein the Fc domains are the Fc structures of IgG Domain.
E56. the antibody or its antigen-binding fragment of embodiment 55, wherein the IgG is IgG1、IgG2、IgG3Or IgG4。
E57. the antibody or its antigen-binding fragment of any one of embodiment 1 to 56, it includes heavy chain, the heavy chain contains SEQ ID NO：42 amino acid sequence.
E58. the antibody or its antigen-binding fragment of any one of embodiment 1 to 56, it includes heavy chain, the heavy chain contains SEQ ID NO：64 amino acid sequence.
E59. the antibody or its antigen-binding fragment of any one of embodiment 1 to 56, it includes heavy chain, the heavy chain contains SEQ ID NO：66 amino acid sequence.
E60. the antibody or its antigen-binding fragment of any one of embodiment 1 to 59, it includes heavy chain, the heavy chain contains SEQ ID NO：37 amino acid sequence.
E61. the antibody or its antigen-binding fragment of any one of embodiment 1 to 60, it includes protected by being present in ATCC The VH sequences of Insert Fragment coding in the plasmid of Tibetan PTA-122329 preservations.
E62. the antibody or its antigen-binding fragment of any one of embodiment 1 to 61, it includes protected by being present in ATCC The VL sequences of Insert Fragment coding in the plasmid of Tibetan PTA-122328 preservations.
E63. the antibody or its antigen-binding fragment of any one of embodiment 1 to 4, wherein the epitope further includes one It is or multiple selected from the residue of the following group：Glu 100, Glu 101, Asp 102, Gly 104 and Tyr 109 are (according to SEQ ID NO：2 numberings).
E64. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 any one, wherein the epitope is further Comprising Glu 100, Glu 101, Asp 102, Gly 104 and Tyr 109 (according to SEQ ID NO：2 numberings).
E65. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 64 any one, wherein the epitope is not It is selected from comprising one or more with the residue of the following group：P103、T111、Y113、F114、N116、Q118、Q121、C122、E123、 R124, F125, K126 and L140 are (according to SEQ ID NO：2 numberings).
E66. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 65 any one, wherein the epitope is not Comprising：P103, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126 and L140 (according to SEQ ID NO：2 numberings).
E67. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 64 any one, wherein the epitope is not It is selected from comprising one or more with the residue of the following group：D31, D32, P34, C35, K36, P103, K126, Y127, G128 are (according to SEQ ID NO：2 numberings).
E68. embodiment 1 to 4,63 to 64 and any one of 67 antibody or its antigen-binding fragment, wherein described Epitope does not include：D31, D32, P34, C35, K36, P103, K126, Y127, G128 are (according to SEQ ID NO：2 numberings).
E69. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 68 any one, it includes following residue (being numbered according to Kabat)：H33Ala、H35Gln、H52Ser、H53Asn、H55 Arg、H56Ser、H95Phe、H96Leu、 H97His, H99Ser, H101Asp, L31Met, L32Tyr, L34His, L36Tyr, L50Arg, L91Trp and L96Tyr.
E70. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 69 any one, it is described it includes VH VH contains：
(a) SEQ ID NO are included：The CDR-H1 of 48 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 49 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 50 amino acid sequence.
E71. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 69 any one, it includes SEQ ID NO：51 CDR-H1, CDR-H2 and CDR-H3 sequence.
E72. embodiment 1 to 4 and any one of 63 to 71 antibody or its antigen-binding fragment, it includes mankind VH3, VH1 or VH5 frame sequences.
E73. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 72 any one, it includes human reproduction It is the VH frame sequences of IGHV3-23 or IGHV1-69.
E74. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 72 any one, it includes human reproduction It is the VH frame sequences of IGHV3-7.
E75. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 71 any one, it includes mankind VH lifes It is shared frame sequence to grow.
E76. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 75 any one, it is described it includes VH VH contains with being selected from at least 90% identical amino acid sequence of the amino acid sequence of the following group：SEQ ID NO：67th, 69,51 and 79。
E77. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 76 any one, it is described it includes VH VH contains selected from the amino acid sequence of the following group：SEQ ID NO：67th, 69,51 and 79.
E78. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 77 any one, it is described it includes VH VH contains SEQ ID NO：67 amino acid sequence.
E79. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 77 any one, it is described it includes VH VH contains SEQ ID NO：69 amino acid sequence.
E80. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 77 any one, it is described it includes VH VH contains SEQ ID NO：51 amino acid sequence.
E81. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 77 any one, it includes weight chain variable Area (VH), the VH contain SEQ ID NO：79 amino acid sequence.
E82. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 81 any one, it is described it includes VL VL contains：
(a) SEQ ID NO are included：The CDR-L1 of 43 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 44 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 45 amino acid sequence.
E83. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 81 any one, it includes SEQ ID NO：46 CDR-L1, CDR-L2 and CDR-L3 sequence.
E84. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 83 any one, it includes mankind VKOr VλFrame sequence.
E85. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 84 any one, it includes human reproduction It is the VL frame sequences of IGKV3-20 or IGKV1-39.
E86. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 83 any one, it includes mankind VL lifes It is shared frame sequence to grow.
E87. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 86 any one, it is described it includes VL VL contains with being selected from at least 90% identical amino acid sequence of the sequence of the following group：SEQ ID NO：46th, 71,73,75 and 77.
E88. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 87 any one, it is described it includes VL VL contains selected from the amino acid sequence of the following group：SEQ ID NO：46th, 71,73,75 and 77.
E89. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 88 any one, it is described it includes VL VL contains SEQ ID NO：46 amino acid sequence.
E90. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 88 any one, it is described it includes VL VL contains SEQ ID NO：71 amino acid sequence.
E91. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 88 any one, it is described it includes VL VL contains SEQ ID NO：73 amino acid sequence.
E92. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 88 any one, it is described it includes VL VL contains SEQ ID NO：75 amino acid sequence.
E93. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 88 any one, it is described it includes VL VL contains SEQ ID NO：77 amino acid sequence.
E94. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 93 any one, it is described it includes CH CH contains and SEQ ID NO：20 at least 90% identical amino acid sequences.
E95. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 94 any one, it is described it includes CH CH contains SEQ ID NO：20 amino acid sequence.
E96. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 95 any one, it is described it includes CL CL contains and SEQ ID NO：26 at least 90% identical amino acid sequences.
E97. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 96 any one, it is described it includes CL CL contains SEQ ID NO：26 amino acid sequence.
E98. the antibody or its antigen-binding fragment of embodiment 1 to 4 and 63 to 97 any one, it includes Fc structures Domain.
E99. the antibody or its antigen-binding fragment of embodiment 98, wherein the Fc domains are the Fc structures of IgA Domain.
E100. the antibody or its antigen-binding fragment of embodiment 99, wherein the IgA is IgA1Or IgA2。
E101. the antibody or its antigen-binding fragment of embodiment 98, wherein the Fc domains be IgD, IgE or The Fc domains of IgM.
E102. the antibody or its antigen-binding fragment of embodiment 98, wherein the Fc domains are the Fc structures of IgG Domain.
E103. the antibody or its antigen-binding fragment of embodiment 102, wherein the IgG is IgG1、IgG2、IgG3Or IgG4。
E104. embodiment 1 to 4 and any one of 63 to 103 antibody or its antigen-binding fragment, it includes heavy chain, The heavy chain contains SEQ ID NO：52 amino acid sequence.
E105. embodiment 1 to 4 and any one of 63 to 103 antibody or its antigen-binding fragment, it includes heavy chain, The heavy chain contains SEQ ID NO：68 amino acid sequence.
E106. embodiment 1 to 4 and any one of 63 to 103 antibody or its antigen-binding fragment, it includes heavy chain, The heavy chain contains SEQ ID NO：70 amino acid sequence.
E107. embodiment 1 to 4 and any one of 63 to 103 antibody or its antigen-binding fragment, it includes heavy chain, The heavy chain contains SEQ ID NO：80 amino acid sequence.
E108. embodiment 1 to 4 and any one of 63 to 107 antibody or its antigen-binding fragment, it includes light chain, The light chain contains SEQ ID NO：47 amino acid sequence.
E109. embodiment 1 to 4 and any one of 63 to 107 antibody or its antigen-binding fragment, it includes light chain, The light chain contains SEQ ID NO：72 amino acid sequence.
E110. embodiment 1 to 4 and any one of 63 to 107 antibody or its antigen-binding fragment, it includes light chain, The light chain contains SEQ ID NO：74 amino acid sequence.
E111. embodiment 1 to 4 and any one of 63 to 107 antibody or its antigen-binding fragment, it includes light chain, The light chain contains SEQ ID NO：76 amino acid sequence.
E112. embodiment 1 to 4 and any one of 63 to 107 antibody or its antigen-binding fragment, it includes light chain, The light chain contains SEQ ID NO：78 amino acid sequence.
E113. epitope in a kind of Kunitz domains 2 (K2) of specific binding tissue factor approach restrainer (TFPI) Separated antibody or its antigen-binding fragment, wherein the epitope include residue Glu101, Pro103, Tyr109, Thr111, Ser119, Gln121, Glu123, Arg124, Lys126 and Leu140 are (according to SEQ ID NO：2 numberings).
E114. the antibody of embodiment 113 or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment are not With reference to the Kunitz domains 1 (K1) of TFPI.
E115. embodiment 113 or 114 antibody or its antigen-binding fragment, wherein the epitope is not comprising one or more It is a to be selected from the residue of the following group：E100, D102, R107, Y113, F114, N116, Q118 and C122 are (according to SEQ ID NO：2 compile Number).
E116. the antibody or its antigen-binding fragment of any one of embodiment 113 to 115, wherein the epitope does not include： E100, D102, R107, Y113, F114, N116, Q118 and C122 are (according to SEQ ID NO：2 numbering).
E117. embodiment 113 or the antibody of 114 or its antigen-binding fragment, wherein the epitope do not include one or It is multiple to be selected from the residue of the following group：D31, D32, P34, C35, K36, E100, I105, R107, G108, Y127 and G128 (according to SEQ ID NO：2 numbering).
E118. the antibody or its antigen-binding fragment of embodiment 113 to 114 and 117 any one, wherein the epitope Do not include：D31, D32, P34, C35, K36, E100, I105, R107, G108, Y127 and G128 are (according to SEQ ID NO：2 volume Number).
E119. the antibody of embodiment 113 to 118 or its antigen-binding fragment, it includes following residue (according to Kabat is numbered)：H50Asp、H57Thr、H58Leu、H59Tyr、H61Gln、H98Asp、H99Tyr、H100Asp、L30His、 L50Trp, L92Tyr, L93Thr, L94Thr and L96Tyr.
E120. the antibody or its antigen-binding fragment of any one of embodiment 113 to 119, it includes VH, the VH contains Have：
(a) SEQ ID NO are included：The CDR-H1 of 87 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 88 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 89 amino acid sequence.
E121. the antibody or its antigen-binding fragment of any one of embodiment 113 to 119, it includes SEQ ID NO：90 CDR-H1, CDR-H2 and CDR-H3 sequence.
E122. the antibody or its antigen-binding fragment of any one of embodiment 113 to 121, it includes the mankind VH3, VH1 or VH5 frame sequences.
E123. the antibody or its antigen-binding fragment of any one of embodiment 113 to 122, it includes human reproduction system The VH frame sequences of IGHV3-23, IGHV1-69 or IGHV3-7.
E124. the antibody or its antigen-binding fragment of any one of embodiment 113 to 121, it includes mankind's VH system genitales Shared frame sequence.
E125. the antibody or its antigen-binding fragment of any one of embodiment 113 to 124, it includes VH, the VH contains With selected from at least 90% identical amino acid sequence of the amino acid sequence of the following group：SEQ ID NO：90、95、97、99、101、 103rd, 105 and 107.
E126. the antibody or its antigen-binding fragment of any one of embodiment 113 to 125, it includes VH, the VH contains Selected from the amino acid sequence of the following group：SEQ ID NO：90th, 95,97,99,101,103,105 and 107.
E127. the antibody or its antigen-binding fragment of any one of embodiment 113 to 126, it includes VH, the VH contains SEQ ID NO：90 amino acid sequence.
E128. the antibody or its antigen-binding fragment of any one of embodiment 113 to 126, it includes VH, the VH contains SEQ ID NO：95 amino acid sequence.
E129. the antibody or its antigen-binding fragment of any one of embodiment 113 to 126, it includes VH, the VH contains SEQ ID NO：97 amino acid sequence.
E130. the antibody or its antigen-binding fragment of any one of embodiment 113 to 126, it includes VH, the VH contains SEQ ID NO：99 amino acid sequence.
E131. the antibody or its antigen-binding fragment of any one of embodiment 113 to 126, it includes VH, the VH contains SEQ ID NO：101 amino acid sequence.
E132. the antibody or its antigen-binding fragment of any one of embodiment 113 to 126, it includes VH, the VH contains SEQ ID NO：103 amino acid sequence.
E133. the antibody or its antigen-binding fragment of any one of embodiment 113 to 126, it includes VH, the VH contains SEQ ID NO：105 amino acid sequence.
E134. the antibody or its antigen-binding fragment of any one of embodiment 113 to 126, it includes VH, the VH contains SEQ ID NO：107 amino acid sequence.
E135. the antibody or its antigen-binding fragment of any one of embodiment 113 to 134, it includes VL, the VL contains Have：
(a) SEQ ID NO are included：The CDR-L1 of 81 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 82 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 83 amino acid sequence.
E136. the antibody or its antigen-binding fragment of any one of embodiment 113 to 134, it includes SEQ ID NO：84 CDR-L1, CDR-L2 and CDR-L3 sequence.
E137. the antibody or its antigen-binding fragment of any one of embodiment 113 to 136, it includes mankind VKOr VλFramework Sequence.
E138. the antibody or its antigen-binding fragment of any one of embodiment 113 to 137, it includes human reproduction system The VL frame sequences of IGKV3-20 or IGKV1-39.
E139. the antibody or its antigen-binding fragment of any one of embodiment 113 to 136, it includes mankind's VL system genitales Shared frame sequence.
E140. the antibody or its antigen-binding fragment of any one of embodiment 113 to 139, it includes VL, the VL contains With selected from at least 90% identical amino acid sequence of the amino acid sequence of the following group：SEQ ID NO：84th, 109 and 111.
E141. the antibody or its antigen-binding fragment of any one of embodiment 113 to 140, it includes VL, the VL contains Selected from the amino acid sequence of the following group：SEQ ID NO：84th, 109 and 111.
E142. the antibody or its antigen-binding fragment of any one of embodiment 113 to 141, it includes VL, the VL contains SEQ ID NO：84 amino acid sequence.
E143. the antibody or its antigen-binding fragment of any one of embodiment 113 to 141, it includes VL, the VL contains SEQ ID NO：109 amino acid sequence.
E144. the antibody or its antigen-binding fragment of any one of embodiment 113 to 141, it includes VL, the VL contains SEQ ID NO：111 amino acid sequence.
E145. the antibody or its antigen-binding fragment of any one of embodiment 113 to 144, it includes CH, the CH contains With SEQ ID NO：20 at least 90% identical amino acid sequences.
E146. the antibody or its antigen-binding fragment of any one of embodiment 113 to 145, it includes CH, the CH contains SEQ ID NO：20 amino acid sequence.
E147. the antibody or its antigen-binding fragment of any one of embodiment 113 to 144, it includes CH, the CH contains With SEQ ID NO：91 at least 90% identical amino acid sequences.
E148. the antibody or its antigen-binding fragment of embodiment 113 to 144 and 147 any one, it includes CH, institute State CH and contain SEQ ID NO：91 amino acid sequence.
E149. the antibody or its antigen-binding fragment of any one of embodiment 113 to 148, it includes CL, the CL contains With SEQ ID NO：14 at least 90% identical amino acid sequences.
E150. the antibody or its antigen-binding fragment of any one of embodiment 113 to 149, it includes CL, the CL contains SEQ ID NO：14 amino acid sequence.
E151. the antibody or its antigen-binding fragment of any one of embodiment 113 to 148, it includes CL, the CL contains With SEQ ID NO：85 at least 90% identical amino acid sequences.
E152. embodiment 113 to 148 and any one of the 151st antibody or its antigen-binding fragment, it includes CL, The CL contains SEQ ID NO：85 amino acid sequence.
E153. the antibody or its antigen-binding fragment of any one of embodiment 113 to 152, it includes Fc domains.
E154. the antibody or its antigen-binding fragment of embodiment 153, wherein the Fc domains be IgA (such as IgA1Or IgA2) Fc domains.
E155. the antibody or its antigen-binding fragment of embodiment 153, wherein the Fc domains be IgD, IgE or The Fc domains of IgM.
E156. the antibody or its antigen-binding fragment of embodiment 153, wherein the Fc domains are the Fc knots of IgG Structure domain.
E157. the antibody or its antigen-binding fragment of any one of embodiment 156, wherein the IgG is IgG1、IgG2、 IgG3Or IgG4。
E158. the antibody or its antigen-binding fragment of any one of embodiment 113 to 157, it includes heavy chain, the heavy chain Contain SEQ ID NO：92 amino acid sequence.
E159. the antibody or its antigen-binding fragment of any one of embodiment 113 to 157, it includes heavy chain, the heavy chain Contain SEQ ID NO：94 amino acid sequence.
E160. the antibody or its antigen-binding fragment of any one of embodiment 113 to 157, it includes heavy chain, the heavy chain Contain SEQ ID NO：96 amino acid sequence.
E161. the antibody or its antigen-binding fragment of any one of embodiment 113 to 157, it includes heavy chain, the heavy chain Contain SEQ ID NO：98 amino acid sequence.
E162. the antibody or its antigen-binding fragment of any one of embodiment 113 to 157, it includes heavy chain, the heavy chain Contain SEQ ID NO：100 amino acid sequence.
E163. the antibody or its antigen-binding fragment of any one of embodiment 113 to 157, it includes heavy chain, the heavy chain Contain SEQ ID NO：102 amino acid sequence.
E164. the antibody or its antigen-binding fragment of any one of embodiment 113 to 157, it includes heavy chain, the heavy chain Contain SEQ ID NO：104 amino acid sequence.
E165. the antibody or its antigen-binding fragment of any one of embodiment 113 to 157, it includes heavy chain, the heavy chain Contain SEQ ID NO：106 amino acid sequence.
E166. the antibody or its antigen-binding fragment of any one of embodiment 113 to 157, it includes heavy chain, the heavy chain Contain SEQ ID NO：108 amino acid sequence.
E167. the antibody or its antigen-binding fragment of any one of embodiment 113 to 166, it includes light chain, the light chain Contain SEQ ID NO：86 amino acid sequence.
E168. the antibody or its antigen-binding fragment of any one of embodiment 113 to 166, it includes light chain, the light chain Contain SEQ ID NO：93 amino acid sequence.
E169. the antibody or its antigen-binding fragment of any one of embodiment 113 to 166, it includes light chain, the light chain Contain SEQ ID NO：110 amino acid sequence.
E170. the antibody or its antigen-binding fragment of any one of embodiment 113 to 166, it includes light chain, the light chain Contain SEQ ID NO：112 amino acid sequence.
E171. a kind of separated antibody or its antigen binding fragment of the Kunitz domains 2 (K2) of specific binding TFPI Section, it includes VH, the VH contains：
(a) SEQ ID NO are included：The CDR-H1 of 16 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 17 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 18 amino acid sequence.
E172. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：19 CDR-H1, CDR-H2 and CDR-H3 sequence.
E173. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes VL, the VL contain：
(a) SEQ ID NO are included：The CDR-L1 of 10 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 11 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 12 amino acid sequence.
E174. the separated antibody or its antigen-binding fragment of the K2 domains of the different combination TFPI of characteristic a kind of, it includes SEQ ID NO：13 CDR-L1, CDR-L2 and CDR-L3 sequence.
El75. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes：
(i) VH, it includes：
(a) SEQ ID NO are included：The CDR-H1 of 16 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 17 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 18 amino acid sequence；
And (ii) VL, it includes：
(a) SEQ ID NO are included：The CDR-L1 of 10 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 11 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 12 amino acid sequence.
E176. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：19 CDR-H1, CDR-H2 and CDR-H3 sequence and SEQ ID NO：13 CDR-L1, CDR L3 and CDR-L3 Sequence.
E177. the antibody or its antigen-binding fragment of any one of embodiment 171 to 176, it includes the mankind VH3, VH1 or VH5 frame sequences.
E178. the antibody or its antigen-binding fragment of any one of embodiment 171 to 177, it includes human reproduction system The VH frame sequences of IGHV3-23, IGHV1-69 or IGHV3-7.
E179. the antibody or its antigen-binding fragment of any one of embodiment 171 to 176, it includes mankind's VH system genitales Shared frame sequence.
E180. the antibody or its antigen-binding fragment of any one of embodiment 171 to 179, it includes VH, the VH contains With SEQ ID NO：19 at least 90% identical amino acid sequences.
E181. the antibody or its antigen-binding fragment of any one of embodiment 171 to 180, it includes VH, the VH contains SEQ ID NO：19 amino acid sequence.
E182. the antibody or its antigen-binding fragment of any one of embodiment 171 to 181, it includes mankind VKOr VλFramework Sequence.
E183. the antibody or its antigen-binding fragment of any one of embodiment 171 to 182, it includes human reproduction system The VL frame sequences of IGKV3-20 or IGKV1-39.
E184. the antibody or its antigen-binding fragment of any one of embodiment 171 to 181, it includes mankind's VL system genitales Shared frame sequence.
E185. the antibody or its antigen-binding fragment of any one of embodiment 171 to 184, it includes VL, the VL contains With SEQ ID NO：13 at least 90% identical amino acid sequences.
E186. the antibody or its antigen-binding fragment of any one of embodiment 171 to 185, it includes VL, the VL contains SEQ ID NO：13 amino acid sequence.
E187. the antibody or its antigen-binding fragment of any one of embodiment 171 to 186, it includes CH, the CH contains With SEQ ID NO：20 at least 90% identical amino acid sequences.
E188. the antibody or its antigen-binding fragment of any one of embodiment 171 to 187, it includes CH, the CH contains SEQ ID NO：20 amino acid sequence.
E189. the antibody or its antigen-binding fragment of any one of embodiment 171 to 188, it includes CL, the CL contains With SEQ ID NO：14 at least 90% identical amino acid sequences.
E190. the antibody or its antigen-binding fragment of any one of embodiment 171 to 189, it includes CL, the CL contains SEQ ID NO：14 amino acid sequence.
E191. the antibody or its antigen-binding fragment of any one of embodiment 171 to 190, it includes Fc domains.
E192. the antibody or its antigen-binding fragment of embodiment 191, wherein the Fc domains be IgA (such as IgA1Or IgA2), the Fc domains of IgD, IgE or IgM.
E193. the antibody or its antigen-binding fragment of embodiment 191, wherein the Fc domains are the Fc knots of IgG Structure domain.
E194. the antibody or its antigen-binding fragment of embodiment 191, wherein the IgG is IgG1、IgG2、IgG3Or IgG4。
E195. the antibody or its antigen-binding fragment of any one of embodiment 171 to 194, it includes heavy chain, the heavy chain Contain SEQ ID NO：21 amino acid sequence.
E196. the antibody or its antigen-binding fragment of any one of embodiment 171 to 195, it includes light chain, the light chain Contain SEQ ID NO：15 amino acid sequence.
E197. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes VH, the VH contain：
(a) SEQ ID NO are included：The CDR-H1 of 28 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 29 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 30 amino acid sequence.
E198. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：31 CDR-H1, CDR-H2 and CDR-H3 sequence.
E199. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes VL, the VL：
(a) SEQ ID NO are included：The CDR-L1 of 22 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 23 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 24 amino acid sequence.
E200. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：25 CDR-L1, CDR-L2 and CDR-L3 sequence.
E201. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes：
(i) VH, it includes：
(a) SEQ ID NO are included：The CDR-H1 of 28 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 29 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 30 amino acid sequence；
And (ii) VL, it includes：
(a) SEQ ID NO are included：The CDR-L1 of 22 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 23 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 24 amino acid sequence.
E202. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：31 CDR-H1, CDR-H2 and CDR-H3 sequence and SEQ ID NO：25 CDR-L1, CDR L2 and CDR-L3 Sequence.
E203. the antibody or its antigen-binding fragment of any one of embodiment 197 to 202, it includes the mankind VH3, VH1 or VH5 frame sequences.
E204. the antibody or its antigen-binding fragment of any one of embodiment 197 to 203, it includes human reproduction system The VH frame sequences of IGHV3-23, IGHV1-69 or IGHV3-7.
E205. the antibody or its antigen-binding fragment of any one of embodiment 197 to 202, it includes mankind's VH system genitales Shared frame sequence.
E206. the antibody or its antigen-binding fragment of any one of embodiment 197 to 205, it includes VH, the VH contains With SEQ ID NO：31 at least 90% identical amino acid sequences.
E207. the antibody or its antigen-binding fragment of any one of embodiment 197 to 206, it includes VH, the VH contains SEQ ID NO：31 amino acid sequence.
E208. the antibody or its antigen-binding fragment of any one of embodiment 197 to 207, it includes mankind VKOr VλFramework Sequence.
E209. the antibody or its antigen-binding fragment of any one of embodiment 197 to 208, it includes human reproduction system The VL frame sequences of IGKV3-20 or IGKV1-39.
E210. the antibody or its antigen-binding fragment of any one of embodiment 197 to 207, it includes mankind's VL system genitales Shared frame sequence.
E211. the antibody or its antigen-binding fragment of any one of embodiment 197 to 210, it includes VL, the VL contains With SEQ ID NO：25 at least 90% identical amino acid sequences.
E212. the antibody or its antigen-binding fragment of any one of embodiment 197 to 211, it includes VL, the VL contains SEQ ID NO：25 amino acid sequence.
E213. the antibody or its antigen-binding fragment of any one of embodiment 197 to 212, it includes VL, the VL contains With SEQ ID NO：20 at least 90% identical amino acid sequences.
E214. the antibody or its antigen-binding fragment of any one of embodiment 197 to 213, it includes VL, the VL contains SEQ ID NO：20 amino acid sequence.
E215. the antibody or its antigen-binding fragment of any one of embodiment 197 to 214, it includes VL, the VL contains With SEQ ID NO：26 at least 90% identical amino acid sequences.
E216. the antibody or its antigen-binding fragment of any one of embodiment 197 to 215, it includes VL, the VL contains SEQ ID NO：26 amino acid sequence.
E217. the antibody or its antigen-binding fragment of any one of embodiment 197 to 216, it includes Fc domains.
E218. the antibody or its antigen-binding fragment of embodiment 217, wherein the Fc domains be IgA (such as IgA1Or IgA2), the Fc domains of IgD, IgE or IgM.
E219. the antibody or its antigen-binding fragment of embodiment 217, wherein the Fc domains be IgG (such as IgG1、IgG2、IgG3Or IgG4) Fc domains.
E220. the antibody or its antigen-binding fragment of any one of embodiment 197 to 219, it includes heavy chain, the heavy chain Contain SEQ ID NO：32 amino acid sequence.
E221. the antibody or its antigen-binding fragment of any one of embodiment 197 to 220, it includes light chain, the light chain Contain SEQ ID NO：27 amino acid sequence.
E222. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes Heavy chain variable region (VH), the VH contain：
(a) SEQ ID NO are included：The CDR-H1 of 58 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 59 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 60 amino acid sequence.
E223. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：61 CDR-H1, CDR-H2 and CDR-H3 sequence.
E224. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes VL, the VL contain：
(a) SEQ ID NO are included：The CDR-L1 of 53 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 54 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 55 amino acid sequence.
E225. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：56 CDR-L1, CDR-L2 and CDR-L3 sequence.
E226. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes：
(i) VH, it includes：
(a) SEQ ID NO are included：The CDR-H1 of 58 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 59 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 60 amino acid sequence；
And (ii) VL, it includes：
(a) SEQ ID NO are included：The CDR-L1 of 53 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 54 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 55 amino acid sequence.
E227. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：61 CDR-H1, CDR-H2 and CDR-H3 sequence and SEQ ID NO：56 CDR-L1, CDR L3 and CDR-L3 Sequence.
E228. the antibody or its antigen-binding fragment of any one of embodiment 222 to 227, it includes the mankind VH3, VH1 or VH5 frame sequences.
E229. the antibody or its antigen-binding fragment of any one of embodiment 222 to 228, it includes human reproduction system The VH frame sequences of IGHV3-23, IGHV1-69 or IGHV3-7.
E230. the antibody or its antigen-binding fragment of any one of embodiment 222 to 227, it includes mankind's VH system genitales Shared frame sequence.
E231. the antibody or its antigen-binding fragment of any one of embodiment 222 to 230, it includes VH, the VH contains With SEQ ID NO：61 at least 90% identical amino acid sequences.
E232. the antibody or its antigen-binding fragment of any one of embodiment 222 to 231, it includes VH, the VH contains SEQ ID NO：61 amino acid sequence.
E233. the antibody or its antigen-binding fragment of any one of embodiment 222 to 232, it includes mankind VKOr VλFramework Sequence.
E234. the antibody or its antigen-binding fragment of any one of embodiment 222 to 233, it includes human reproduction system IGKV3-20 or IGKV1-39 frame sequences.
E235. the antibody or its antigen-binding fragment of any one of embodiment 222 to 232, it includes mankind's VL system genitales Shared frame sequence.
E236. the antibody or its antigen-binding fragment of any one of embodiment 222 to 235, it includes VL, the VL contains With SEQ ID NO：56 at least 90% identical amino acid sequences.
E237. the antibody or its antigen-binding fragment of any one of embodiment 222 to 236, it includes VL, the VL contains SEQ ID NO：56 amino acid sequence.
E238. the antibody or its antigen-binding fragment of any one of embodiment 222 to 237, it includes CH, the CH contains With SEQ ID NO：20 at least 90% identical amino acid sequences.
E239. the antibody or its antigen-binding fragment of any one of embodiment 222 to 238, it includes CH, the CH contains SEQ ID NO：20 amino acid sequence.
E240. the antibody or its antigen-binding fragment of any one of embodiment 222 to 239, it includes CL, the CL contains With SEQ ID NO：26 at least 90% identical amino acid sequences.
E241. the antibody or its antigen-binding fragment of any one of embodiment 222 to 240, it includes CL, the CL contains SEQ ID NO：26 amino acid sequence.
E242. the antibody or its antigen-binding fragment of any one of embodiment 222 to 241, it includes Fc domains.
E243. the antibody or its antigen-binding fragment of any one of embodiment 222 to 242, wherein the Fc domains are IgA (such as IgA1Or IgA2), the Fc domains of IgD, IgE or IgM.
E244. the antibody or its antigen-binding fragment of embodiment 242, wherein the Fc domains be IgG (such as gG1、IgG2、IgG3Or IgG4) Fc domains.
E245. the antibody or its antigen-binding fragment of any one of embodiment 222 to 244, it includes contain SEQ ID NO：62 amino acid sequence.
E246. the antibody or its antigen-binding fragment of any one of embodiment 222 to 245, it includes contain SEQ ID NO：57 amino acid sequence.
E247. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes VH, the VH contain：
(a) SEQ ID NO are included：The CDR-H1 of 118 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 119 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 120 amino acid sequence.
E248. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：121 CDR-H1, CDR-H2 and CDR-H3 sequence.
E249. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes VL, the VL contain：
(a) SEQ ID NO are included：The CDR-L1 of 113 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 114 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 115 amino acid sequence.
E250. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：116 CDR-L1, CDR-L2BCDR-L3 sequence.
E251. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes：
(i) VH, it includes：
(a) SEQ ID NO are included：The CDR-H1 of 118 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 119 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 120 amino acid sequence；
And (ii) VL, it includes：
(a) SEQ ID NO are included：The CDR-L1 of 113 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 114 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 115 amino acid sequence.
E252. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：121 CDR-H1, CDR-H2 and CDR-H3 sequence, and SEQID NO：116 CDR-L1, CDR-L2 and CDR-L3 sequences.
E253. the antibody or its antigen-binding fragment of any one of embodiment 247 to 253, it includes the mankind VH3, VH1 or VH5 frame sequences.
E254. the antibody or its antigen-binding fragment of any one of embodiment 247 to 253, it includes human reproduction system The VH frame sequences of IGHV3-23, IGHV1-69 or IGHV3-7.
E255. the antibody or its antigen-binding fragment of any one of embodiment 247 to 252, it includes mankind's VH system genitales Shared frame sequence.
E256. the antibody or its antigen-binding fragment of any one of embodiment 247 to 255, it includes VH, the VH contains With SEQ ID NO：121 at least 90% identical amino acid sequences.
E257. the antibody or its antigen-binding fragment of any one of embodiment 247 to 256, it includes VH, the VH contains SEQ ID NO：121 amino acid sequence.
E258. the antibody or its antigen-binding fragment of any one of embodiment 247 to 257, it includes mankind VKOr VλFramework Sequence.
E259. the antibody or its antigen-binding fragment of any one of embodiment 247 to 258, it includes human reproduction system IGKV3-20 or IGKV1-39 frame sequences.
E260. the antibody or its antigen-binding fragment of any one of embodiment 247 to 257, it includes mankind's VL system genitales Shared frame sequence.
E261. the antibody or its antigen-binding fragment of any one of embodiment 247 to 260, it includes VL, the VL contains With SEQ ID NO：116 at least 90% identical amino acid sequences.
E262. the antibody or its antigen-binding fragment of any one of embodiment 247 to 261, it includes VL, the VL contains SEQ ID NO：116 amino acid sequence.
E263. the antibody or its antigen-binding fragment of any one of embodiment 247 to 262, it includes CH, the CH contains With SEQ ID NO：91 at least 90% identical amino acid sequences.
E264. the antibody or its antigen-binding fragment of any one of embodiment 247 to 263, it includes CH, the CH contains SEQ ID NO：91 amino acid sequence.
E265. the antibody or its antigen-binding fragment of any one of embodiment 247 to 264, it includes CL, the CL contains With SEQ ID NO：85 at least 90% identical amino acid sequences.
E266. the antibody or its antigen-binding fragment of any one of embodiment 247 to 265, it includes CL, the CL contains SEQ ID NO：85 amino acid sequence.
E267. the antibody or its antigen-binding fragment of any one of embodiment 247 to 266, it includes Fc domains.
E268. the antibody or its antigen-binding fragment of embodiment 267, wherein the Fc domains be IgA (such as IgA1Or IgA2), the Fc domains of IgD, IgE or IgM.
E269. the antibody or its antigen-binding fragment of embodiment 267, wherein the Fc domains be IgG (such as gG1、IgG2、IgG3Or IgG4) Fc domains.
E270. the antibody or its antigen-binding fragment of any one of embodiment 247 to 269, it includes heavy chain, the heavy chain Contain SEQ ID NO：122 amino acid sequence.
E271. the antibody or its antigen-binding fragment of any one of embodiment 247 to 270, it includes light chain, the light chain Contain SEQ ID NO：117 amino acid sequence.
E272. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes VH, the VH contain：
(a) SEQ ID NO are included：The CDR-H1 of 128 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 129 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 130 amino acid sequence.
E273. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：131 CDR-H1, CDR-H2 and CDR-H3 sequence.
E274. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes VL, the VL contain following group：
(a) SEQ ID NO are included：The CDR-L1 of 123 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 124 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 125 amino acid sequence.
E275. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：126 CDR-L1, CDR L3 and CDR-L3 sequences.
E276. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes：
(i) VH, it includes：
(a) SEQ ID NO are included：The CDR-H1 of 128 amino acid sequence；
(b) SEQ ID NO are included：The CDR-H2 of 129 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-H3 of 130 amino acid sequence；
And (ii) VL, it includes：
(a) SEQ ID NO are included：The CDR-L1 of 123 amino acid sequence；
(b) SEQ ID NO are included：The CDR-L2 of 124 amino acid sequence；And
(c) SEQ ID NO are included：The CDR-L3 of 125 amino acid sequence.
E277. a kind of separated antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, it includes SEQ ID NO：131 CDR-H1, CDR-H2 and CDR-H3 sequence and SEQ ID NO：126 CDR-L1, CDR L3 and CDR- L3 sequences.
E278. the antibody or its antigen-binding fragment of any one of embodiment 272 to 277, it includes the mankind VH3, VH1 or VH5 frame sequences.
E279. the antibody or its antigen-binding fragment of any one of embodiment 272 to 278, it includes mankind IGHV3-23, The VH frame sequences of IGHV1-69 or IGHV3-7.
E280. the antibody or its antigen-binding fragment of any one of embodiment 272 to 277, it includes mankind's VH system genitales Shared frame sequence.
E281. the antibody or its antigen-binding fragment of any one of embodiment 272 to 280, it includes VH, the VH contains With SEQ ID NO：131 at least 90% identical amino acid sequences.
E282. the antibody or its antigen-binding fragment of any one of embodiment 272 to 281, it includes VH, the VH contains SEQ ID NO：131 amino acid sequence.
E283. the antibody or its antigen-binding fragment of any one of embodiment 272 to 282, it includes mankind VKOr VλFramework Sequence.
E284. the antibody or its antigen-binding fragment of any one of embodiment 272 to 283, it includes human reproduction system IGKV3-20 or IGKV1-39 frame sequences.
E285. the antibody or its antigen-binding fragment of any one of embodiment 272 to 282, it includes mankind's VL system genitales Shared frame sequence.
E286. the antibody or its antigen-binding fragment of any one of embodiment 272 to 285, it includes VL, the VL contains With SEQ ID NO：126 at least 90% identical amino acid sequences.
E287. the antibody or its antigen-binding fragment of any one of embodiment 272 to 286, it includes VL, the VL contains SEQ ID NO：126 amino acid sequence.
E288. the antibody or its antigen-binding fragment of any one of embodiment 272 to 287, it includes CH, the CH contains With SEQ ID NO：91 at least 90% identical amino acid sequences.
E289. the antibody or its antigen-binding fragment of any one of embodiment 272 to 288, it includes CH, the CH contains SEQ ID NO：91 amino acid sequence.
E290. the antibody or its antigen-binding fragment of any one of embodiment 272 to 289, it wraps CL, and the CL contains With SEQ ID NO：85 at least 90% identical amino acid sequences.
E291. the antibody or its antigen-binding fragment of any one of embodiment 272 to 290, it includes CL, the CL contains SEQ ID NO：85 amino acid sequence.
E292. the antibody or its antigen-binding fragment of any one of embodiment 272 to 291, it includes Fc domains.
E293. the antibody or its antigen-binding fragment of embodiment 292, wherein the Fc domains be IgA (such as IgA1Or IgA2), the Fc domains of IgD, IgE or IgM.
E294. the antibody or its antigen-binding fragment of embodiment 292, wherein the Fc domains be IgG (such as IgG1、IgG2、IgG3Or IgG4) Fc domains.
E295. the antibody or its antigen-binding fragment of any one of embodiment 272 to 294, it includes heavy chain, the heavy chain Contain SEQ ID NO：132 amino acid sequence.
E296. the antibody or its antigen-binding fragment of any one of embodiment 272 to 295, it includes light chain, the light chain Contain SEQ ID NO：127 amino acid sequence.
E297. a kind of antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, wherein the antibody Or its antigen-binding fragment and the antibody or its antigen-binding fragment competition binding TFPI of any one of embodiment 1 to 296.
E298. a kind of antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, wherein the antibody Or its antigen-binding fragment is with being selected from the antibody competition combination TFPI of the following group：TFPI-3、TFPI-21、TFPI-23、TFPI- 24、TFPI-26、TFPI-106、TFPI-107、TFPI-108、TFPI-109、TFPI-110、TFPI-111、TFPI-112、 TFPI-113、TFPI-114、TFPI-115、TFPI-118、TFPI-119、TFPI-122、TFPI-123、TFPI-126、 4D8.b1, mu-hu 4D8 chimeras, 4D8-Vk1.0x VH1.0,4D8-Vk1.0x VH1.1,4D8-Vk1.0x VH1.2, 4D8-Vk1.0x VH1.3、4D8-Vk1.0x VH1.4、4D8-Vk1.0x VH1.5、4D8-Vk1.0x VH1.6、4D8- Vk1.1x VH1.0、4D8-Vk1.1x VH1.1、4D8-Vk1.1x VH1.2、4D8-Vk1.1x VH1.3、4D8-Vk1.1x VH1.4,4D8-Vk1.1x VH1.5,4D8-Vk1.1x VH1.6, hz4D8,6B7.c5 and 7A4.D9.
E299. a kind of antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, wherein the antibody Or its antigen-binding fragment is with being selected from the antibody competition combination TFPI of the following group：TFPI-23, TFPI-24, TFPI-106 and TFPI-118。
E300. the antibody or its antigen-binding fragment of embodiment 299, wherein the antibody or its antigen-binding fragment With TFPI-23 or TFPI-106 competition bindings TFPI.
E301. the antibody or its antigen-binding fragment of embodiment 299, wherein the antibody or its antigen-binding fragment With TFPI-24 or TFPI-118 competition bindings TFPI.
E302. a kind of antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, wherein the antibody Or its antigen-binding fragment and antibody 4D8 competition bindings TFPI.
E303. a kind of antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, wherein the antibody Or the antibody of any one of its antigen-binding fragment and embodiment the 1st to 296 or its antigen-binding fragment be bound to it is identical TFPI epitopes.
E304. a kind of antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, wherein the antibody Or its antigen-binding fragment is with being selected from the antibody binding of the following group to identical TFPI epitopes：TFPI-3、TFPI-21、TFPI- 23、TFPI-24、TFPI-26、TFPI-106、TFPI-107、TFPI-108、TFPI-109、TFPI-110、TFPI-111、 TFPI-112、TFPI-113、TFPI-114、TFPI-115、TFPI-118、TFPI-119、TFPI-122、TFPI-123、TFPI- 126th, 4D8.b1, mu-hu 4D8 chimeras, 4D8-Vk1.0x VH1.0,4D8-Vk1.0x VH1.1,4D8-Vk1.0x VH1.2、4D8-Vk1.0x VH1.3、4D8-Vk1.0x VH1.4、4D8-Vk1.0x VH1.5、4D8-Vk1.0x VH1.6、 4D8-Vk1.1x VH1.0、4D8-Vk1.1x VH1.1、4D8-Vk1.1x VH1.2、4D8-Vk1.1x VH1.3、4D8- Vk1.1x VH1.4,4D8-Vk1.1x VH1.5,4D8-Vk1.1x VH1.6, hz4D8,6B7.c5 and 7A4.D9.
E305. a kind of antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, wherein the antibody Or its antigen-binding fragment is with being selected from the antibody binding of the following group to identical TFPI epitopes：TFPI-23、TFPI-24、TFPI- 106 and TFPI-118.
E306. the antibody or its antigen-binding fragment of embodiment 305, wherein the antibody or its antigen-binding fragment Identical TFPI epitopes are bound to TFPI-23 or TFPI-106.
E307. the antibody or its antigen-binding fragment of embodiment 305, wherein the antibody or its antigen-binding fragment Identical TFPI epitopes are bound to TFPI-24 or TFPI-118.
E308. a kind of antibody or its antigen-binding fragment of the K2 domains of specific binding TFPI, wherein the antibody Or its antigen-binding fragment is bound to identical TFPI epitopes with antibody 4D8.
E309. the antibody or its antigen-binding fragment of any one of embodiment 297 to 308, wherein the antibody or it is anti- Former binding fragment is not combined with the K1 domains of TFPI.
E310. the antibody or its antigen-binding fragment of any one of embodiment 1 to 309, wherein the antibody or its antigen Binding fragment be Fc fusion proteins, monoclonal antibody (monobody), big antibody (maxibody), bifunctional antibody, scFab, scFv, Peptibody (peptibody) or foregoing any antigen-binding fragment.
E311. the antibody or its antigen-binding fragment of any one of embodiment 1 to 310, wherein the antibody or its antigen Binding fragment is with about 1 × 10-7M to about 1 × 10-12Binding affinity (Kd) the value combination TFPI of M.
E312. the antibody or its antigen-binding fragment of any one of embodiment 1 to 311, wherein the antibody or its antigen Binding fragment is with about 5 × 10-7M to about 5 × 10-11Binding affinity (Kd) the value combination TFPI of M.
E313. the antibody or its antigen-binding fragment of any one of embodiment 1 to 312, wherein the antibody or its antigen Binding fragment is with about 1 × 10-8M to about 1 × 10-10Binding affinity (Kd) the value combination TFPI of M.
E314. the antibody or its antigen-binding fragment of any one of embodiment 1 to 313, wherein the antibody or its antigen Binding fragment：(i) the measured clotting time in dilution prothrombin time (dPT) measure based on blood plasma is reduced；(ii) Reduce by Thrombectomy (thromboelastrography) or rotation thrombus elasticity measure detection (thromboelastometry) whole blood coagulation time measured by；(iii) fibrin ferment generation is increased；(iv) in the presence of TFPI Increase FXa activity；Or (v) its any combinations.
E315. the antibody or its antigen-binding fragment of embodiment 314, wherein the antibody or its antigen-binding fragment Reduce the measured clotting time in the dilution prothrombin time based on blood plasma.
E316. the antibody or its antigen-binding fragment of embodiment 315, wherein described in the dilution blood coagulation based on blood plasma It is dose dependent that the measured clotting time, which is reduced, in zymogen time measure.
E317. the antibody or its antigen-binding fragment of embodiment 314, wherein the antibody or its antigen-binding fragment Reduce by Thrombectomy or rotate the measured whole blood coagulation time of thrombus elasticity measure detection.
E318. the antibody or its antigen-binding fragment of embodiment 317, wherein described by Thrombectomy or rotation It is dose dependent that the measured whole blood coagulation time of thrombus elasticity measure detection, which is reduced,.
E319. the antibody or its antigen-binding fragment of embodiment 314, wherein the antibody or antigen-binding fragment increase Fibrin ferment is added to generate.
E320. the antibody or its antigen-binding fragment of embodiment 319, wherein the increase of fibrin ferment generation is agent Measure dependence.
E321. the antibody or its antigen-binding fragment of embodiment 314, wherein the antibody or antigen-binding fragment exist Increase FXa activity in the presence of TFPI.
E322. the antibody or its antigen-binding fragment of embodiment 321, wherein described increased in the presence of TFPI FXa activity is dose dependent.
E323. the antibody or its antigen-binding fragment of embodiment 322, wherein the antibody strengthens in the presence of TFPI Platelet accumulation.
E324. the antibody or its antigen-binding fragment of embodiment 323, wherein the enhancing blood in the presence of TFPI is small Plate accumulation is dose dependent.
E325. the antibody or its antigen-binding fragment of embodiment 324, wherein the antibody increases in the presence of TFPI Fibrin generates.
E326. the antibody or its antigen-binding fragment of embodiment 325, wherein described increase fiber in the presence of TFPI Albumen generation is dose dependent.
E327. the antibody or its antigen-binding fragment of embodiment 314, wherein it is to make that the whole blood coagulation time, which is reduced, With the whole blood determination derived from the human patients with heavy hemophilia A.
E328. the antibody or its antigen-binding fragment of embodiment 314, wherein it is to make that the whole blood coagulation time, which is reduced, Measured with the whole blood derived from the human patients with heavy hemophilia A and for the inhibiting antibody of human Factor VIII.
E329. the antibody or its antigen-binding fragment of embodiment 314, wherein it is to make that the whole blood coagulation time, which is reduced, With the whole blood determination derived from the human patients with osculant hemophilia A.
E330. the antibody or its antigen-binding fragment of embodiment 314, wherein it is to make that the whole blood coagulation time, which is reduced, With the whole blood determination derived from the human patients with heavy hemophilia B.
E331. the antibody or its antigen-binding fragment of embodiment 314, wherein it is to make that the whole blood coagulation time, which is reduced, Measured with the whole blood derived from the human patients with heavy hemophilia B and for the inhibiting antibody of human Factor Ⅸ.
E332. the antibody or its antigen-binding fragment of embodiment 314, wherein it is to make that the whole blood coagulation time, which is reduced, With the whole blood determination derived from the human patients with osculant hemophilia B.
E333. the antibody or its antigen-binding fragment of embodiment 314, wherein described measured in dPT measure It is to use the determination of plasma for deriving from the human patients with heavy hemophilia A that clotting time, which is reduced,.
E334. the antibody or its antigen-binding fragment of embodiment 314, wherein described measured in dPT measure It is to use the blood plasma for deriving from the human patients with heavy hemophilia A and the suppression for human Factor VIII that clotting time, which is reduced, Property antibody determination.
E335. the antibody or its antigen-binding fragment of embodiment 314, wherein described measured in dPT measure It is to use the determination of plasma for deriving from the human patients with osculant hemophilia A that clotting time, which is reduced,.
E336. the antibody or its antigen-binding fragment of embodiment 314, wherein described measured in dPT measure It is to use the determination of plasma for deriving from the human patients with heavy hemophilia B that clotting time, which is reduced,.
E337. the antibody or its antigen-binding fragment of embodiment 314, wherein described measured in dPT measure It is to use the blood plasma for deriving from the human patients with heavy hemophilia B and the inhibition for human Factor Ⅸ that clotting time, which is reduced, Antibody determination.
E338. the antibody or its antigen-binding fragment of embodiment 314, wherein described measured in dPT measure It is to use the determination of plasma for deriving from the human patients with osculant hemophilia B that clotting time, which is reduced,.
E339. the antibody or its antigen-binding fragment of embodiment 314, wherein fibrin ferment generation increase is to use Determination of plasma derived from the human patients with heavy hemophilia A.
E340. the antibody or its antigen-binding fragment of embodiment 314, wherein fibrin ferment generation increase is to use What blood plasma derived from the human patients with heavy hemophilia A and the inhibiting antibody for human Factor VIII measured.
E341. the antibody or its antigen-binding fragment of embodiment 314, wherein fibrin ferment generation increase is to use Determination of plasma derived from the human patients with osculant hemophilia A.
E342. the antibody or its antigen-binding fragment of embodiment 314, wherein fibrin ferment generation increase is to use Determination of plasma derived from the human patients with heavy hemophilia B.
E343. the antibody or its antigen-binding fragment of embodiment 314, wherein fibrin ferment generation increase is to use What blood plasma derived from the human patients with heavy hemophilia B and the inhibiting antibody for human Factor Ⅸ measured.
E344. the antibody or its antigen-binding fragment of embodiment 314, wherein fibrin ferment generation increase is to use Determination of plasma derived from the human patients with osculant hemophilia type.
E345. the antibody or its antigen-binding fragment of embodiment 314, antibody described in wherein 100nM or its antigen knot Fragment is closed on the clotting time for reducing the whole blood for deriving from the human patients with heavy hemophilia A at least with being enough to obtain 5% The amount of the recombinant human Factor IX of normal coagulation activity is equally effective.
E346. the antibody or its antigen-binding fragment of embodiment 314, antibody described in wherein 100nM or its antigen knot Close fragment derived from increase the human patients with heavy hemophilia A platelet rich plasma the generation of peak value fibrin ferment up to The amount of few recombinant human Factor IX with being enough to obtain 5% normal coagulation activity is same effective.
E347. the antibody or its antigen-binding fragment of any one of embodiment 1 to 346, wherein the TFPI is the mankind TFPI。
E348. the antibody or its antigen-binding fragment of any one of embodiment 1 to 347, wherein the TFPI includes SEQ ID NO：2 residue 91 to 147.
E349. a kind of separated nucleic acid molecules or nucleic acid molecules class, it includes one or more coding embodiments 1 to Any one of 348 antibody or the nucleotide sequence of its antigen-binding fragment.
E350. a kind of separated nucleic acid molecules for the antibody or its antigen-binding fragment for encoding specific binding TFPI, its Described in nucleic acid include be selected from the nucleotide sequence of the following group：SEQ ID NO：175 nucleotide sequence, SEQ ID NO：176 core Acid sequence, SEQ ID NO：177 nucleotide sequence, SEQ ID NO：178 nucleotide sequence, with ATCC preserving numbers PTA-122328 The Insert Fragment nucleotide sequence of the mAb-TFPI-106 VL carriers of preservation and with ATCC preserving number PTA-122329 preservations The Insert Fragment nucleotide sequence of mAb-TFPI-106VH carriers.
E351. a kind of carrier, it includes the nucleic acid molecules of embodiment 349 and 350.
E352. a kind of host cell, it includes the nucleic acid molecules of embodiment 349 or 350 or the load of embodiment 351 Body.
E353. the host cell of embodiment 352, wherein the cell is mammalian cell.
E354. the host cell of embodiment 353, wherein the host cell be Chinese hamster ovary celI, HEK-293 cells or Sp2.0 cells.
E355. a kind of to prepare antibody or the method for its antigen-binding fragment, it includes the antibody wherein or its antigen Binding fragment is by cultivating the host cell of any one of embodiment 352 to 354 under conditions of the host cell expression.
E356. the method for embodiment 355, it, which is further included, separates the antibody or its antigen-binding fragment.
E357. a kind of antibody or its antigen-binding fragment, it is obtained by embodiment 355 or the method for 356.
E358. a kind of pharmaceutical composition, it includes the antibody or its antigen knot of embodiment 1 to 347 and 357 any one Close fragment and pharmaceutically acceptable carrier (carrier) or excipient.
E359. a kind of active method for reducing tissue factor approach restrainer (TFPI), it includes what is needed to there is this Object applies embodiment 1 to 347 and antibody or its antigen-binding fragment or the embodiment party of 357 any one of therapeutically effective amount The pharmaceutical composition that case is 358.
E360. a kind of method for shortening the bleeding time, it includes the reality that the object needed to there is this applies therapeutically effective amount Apply the antibody or the pharmaceutical composition of its antigen-binding fragment or embodiment 358 of scheme 1 to 347 and 357 any one.
E361. embodiment 358 or the method for 360, wherein the object is the mankind.
E362. the method for any one of embodiment 359 to 361, wherein the object suffers from or easily suffer from blood clotting Lack.
E363. the method for any one of embodiment 359 to 361, wherein the object suffers from or easily different with blood platelet Often.
E364. the method for any one of embodiment 359 to 361, wherein the object suffers from or easily suffer from Hemophilia A and B Or C.
E365. the method for any one of embodiment 359 to 361, wherein the object suffer from or easily with hemophilia A or B。
E366. the method for any one of embodiment 359 to 361, wherein the object suffers from or easily suffer from vascular blood Friendly disease (von Willebrand disease, vWD).
E367. the method for embodiment 360, it further includes the FVII using therapeutically effective amount.
E368. the method for embodiment 367, wherein the method increase fibrin ferment generation in the presence of TFPI.
E369. the method for any one of embodiment 359 to 368, it includes antibody described in intravenous administration or its antigen binding Fragment or pharmaceutical composition.
E370. the method for any one of embodiment 359 to 368, it includes antibody described in subcutaneous administration or its antigen binding Fragment or pharmaceutical composition.
E371. the method for any one of embodiment 359 to 368, wherein the antibody or its antigen-binding fragment or medicine Composition is to apply once within every 3 days, apply once within every 4 days, applying once within every 5 days, apply once within every 6 days, apply once weekly Or apply weekly twice.
E372. the antibody or its antigen-binding fragment or embodiment 358 of embodiment 1 to 347 and 357 any one Pharmaceutical composition, used as medicine.
E373. the antibody or its antigen-binding fragment or embodiment 358 of embodiment 1 to 347 and 357 any one Pharmaceutical composition, be for reduce object TFPI activity.
E374. the antibody or its antigen-binding fragment or embodiment 358 of embodiment 1 to 347 and 357 any one Pharmaceutical composition, be the bleeding time for shortening object.
E375. the antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiment 372 to 374, wherein described Object is the mankind.
E376. the antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiment 372 to 375, wherein described Object is suffered from or easily lacked with blood clotting.
E377. the antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiment 372 to 375, wherein described Object suffers from or easily suffers from Hemophilia A and B or C.
E378. the antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiment 372 to 375, wherein described Object suffers from or easily suffers from hemophilia A or B.
E379. the antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiment 372 to 375, wherein described Object suffers from or easily suffers from von Willebrand disease (vWD).
E380. the antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiment 372 to 375, wherein described Object suffers from or easily suffers from blood platelet disorders.
E381. a kind of embodiment 1 to 348 and any one of 357 antibody or its antigen-binding fragment or embodiment Purposes of the pharmaceutical composition of 358 to the TFPI activity of reduction object.
E382. a kind of embodiment 1 to 348 and any one of 357 antibody or its antigen-binding fragment or embodiment Purposes of the pharmaceutical composition of 358 in the medicine for preparing the TFPI activity for being used to reduce object.
E383. a kind of embodiment 1 to 348 and any one of 357 antibody or its antigen-binding fragment or embodiment Purposes of the pharmaceutical composition of 358 in the bleeding time for shortening object.
E384. a kind of embodiment 1 to 348 and any one of 357 antibody or its antigen-binding fragment or embodiment Purposes of the pharmaceutical composition of 358 in the medicine for preparing the bleeding time for being used to shorten object.
E385. the purposes of any one of embodiment 381 to 384, wherein the object is the mankind.
E386. the purposes of any one of embodiment 381 to 384, wherein the object suffers from or easily suffer from blood clotting Lack.
E387. the purposes of any one of embodiment 381 to 384, wherein the object suffers from or easily suffer from Hemophilia A and B Or C.
E388. the purposes of any one of embodiment 381 to 384, wherein the object suffer from or easily with hemophilia A or B。
E389. the purposes of any one of embodiment 381 to 384, wherein the object suffers from or easily suffer from vascular blood Friendly disease (vWD).
E390. the purposes of any one of embodiment 381 to 384, wherein the object suffers from or easily different with blood platelet Often.
Brief description of the drawings
The eutectic structure of the various anti-TFPI antibody of graphical display of Figure 1A to 1F and the K2 domains of TFPI.Specifically, As shown in fig. 1F, compared with other reference antibodies, exemplary antibodies TFPI-23, TFPI-24 disclosed by the invention and 4D8 are whole It is bound to the non-overlapping epitopes of K2 domains.TFPI-106 and TFPI-23 are bound to same site, and TFPI-118 and TFPI- 24 are bound to same site." R＆D " or " R＆D Fab " refer to the antibody Mab 2974 from R＆D systems.Novo2021 antibody is also Referred to as " hz4F36 "." clone 23 " refers to TFPI-23；" clone 24 " refers to TFPI-24.
The epitope residues being diagrammatically shown in the K2 domains of TFPI of Fig. 2A to 2E with from various anti-TFPI antibody Interaction between paratope residue." R＆D " or " R＆D Fab " refer to the antibody Mab 2974 from R＆D systems. " clone 23 " refers to TFPI-23；" clone 24 " refers to TFPI-24.
Fig. 3 A to 3E are shown in the internal effect of the various anti-TFPI antibody in mouse damage model.Fig. 3 A and 3B, which are shown, to be worked as During using 2A8-200 and 2A8 antibody (being used as in this research and refer to antibody), hemophilia A Factor IX shortage (FVIII-/-) small The duration of mouse bleeding reduction.Fig. 3 C are shown to be resisted when using TFPI 4D8 (control), TFPI-21, TFPI-23 and TFPI-24 During body, the duration of hemophilia A mouse antibodies effect.Fig. 3 D and 3E, which show to work as, applies TFPI-106 and TFPI-118 antibody When, the hemophilia A mouse duration.Specified time point (hour (h)) before damage is logical to hemophilia A mouse (FVIII-/-) Intravenous injection is crossed with 6mg/kg administration of antibodies.Then after afterbody is cross-section, the cumulative volume (μ L) lost blood is measured.Supporting agent (vehicle) the hemophilia A mouse of (brine) processing is as control.All measurements are presented with average value ± SEM.*=P< 0.05.FVIII+ /+(wild type) mouse receives brine.N=5/ groups.
Fig. 4 figures show when apply TFPI-106 antibody when, bleeding duration of the factor Ⅸ deficient mouse after afterbody is cross-section. Specified time point (hour (h)) before damage is to factor Ⅸ deficient mouse by being injected intravenously with 6mg/kg administration of antibodies.Then exist After afterbody is cross-section, the cumulative volume (μ L) lost blood is measured.The hemophilia A mouse of supporting agent (brine) processing is as control.All measurements Presented with average value ± SEM.*=P<0.05.N=4-5/ groups.
Fig. 5 includes A groups and B groups, it respectively includes six groups (Fig. 5 A1 to 6 and Fig. 5 B1 to 6), shows intravital microscopy (IVM) microphoto, it was demonstrated that TFPI platelet thrombuses of vascular injury site and along endothelium in wild-type mice body Detect.Damage of Fig. 5 A displays detected by using the CD42c with reference to the marks of Dylight 649 of the GP1b β on blood platelet The platelet thrombus increase at position.Blood platelet is by organizing 1 (0 second)；2 (15 seconds) of group；3 (30 seconds) of group；Group 4 (60 Second)；5 (90 seconds) of group；And the fluorescence signal detected in 6 (120 seconds) of group proves.Also the negative right of the marks of Alexa 488 is applied According to IgG and and it is not detected by fluorescence.Fig. 5 B comprising group 1 to 6 show the microphoto of IVM, it is proved in wild-type mice After middle induced with laser injury of blood vessel, the platelet thrombus of mouse and there are TFPI along endothelium.0 second (Fig. 5 B, group 1) when not Detect the TFPI (green is shown with grey) that Alexa 488 is marked, and (Fig. 5 B, can see micro- when organizing 2) at 15 seconds Weak signal.At 30 seconds, (Fig. 5 B, green florescent signal increased and faint danger signal (Dylight 649 can be seen when organizing 3) The CD42c of mark), it is indicated about with detecting that the same loci of TFPI detects platelet accumulation.Fig. 5 B groups 4 (60 seconds) are aobvious Show that (the two is light gray, and wherein red fluorescence can be seen on the left of vascular lesions sites for strong green and red fluorescent Arrive, and green is mainly towards detecting on the right side of damage location), it proves to detect platelet accumulation in damage location And TFPI.Fig. 5 B groups 5 are shown in 60 seconds red (blood platelet) in damage location and green (TFPI) fluorescence signal, wherein this Signal of two kinds of signals all compared with 30 seconds is strong.Fig. 5 B groups 6 show that red (blood platelet) signal and green (TFPI) signal weaken, Still the two can be detected at 120 seconds in damage location.
The figure of Fig. 6 A show using IVM assessment in hemophilia A mouse after induced with laser injury of blood vessel TFPI-106 only The amount of blood effect, the wherein platelet thrombus is to represent (*=P with area under the curve (AUC)<0.005 is noted).Fig. 6 A are shown Compared with hemophilia A mouse with applying brine lacks platelet accumulation when 0.5 is small, only receive the wild-type mice of saline control (WT) after injury 0.5 it is small when damage location platelet accumulation.Wherein administered recombinant Factor IX (rFVIII) or TFPI- 106 hemophilia A mouse detects platelet accumulation when 0.5 is small.Using the hemophilia A mouse of TFPI-106 when 168 is small Remain to detect thrombus accumulation.
The figure of Fig. 6 B show using IVM assessment in hemophilia A mouse after induced with laser injury of blood vessel TFPI-106 only Blood effect, wherein the fibrin growing amount are to represent (*=P with area under the curve (AUC)<0.005 is noted).Fig. 6 B are shown Hemophilia A mouse with applying brine lacks detectable fibrin when 0.5 is small and generates, and only receives the wild of saline control Type mouse (WT) after injury 0.5 it is small when damage location fibrin generation.Wherein administered recombinant Factor IX (rFVIII) or TFPI-106 hemophilia A mouse can detect when 0.5 is small fibrin generation.Using the blood of TFPI-106 Friendly disease A mouse remain to detect that fibrin is generated when 168 is small.
The figure that Fig. 7 describes is shown in the presence of 1pm tissue factors and 4 μM of phosphatide, is applied in heavy hemophilia A plasma TFPI-106 and recombinant factor VIIa (rFVIIa) generates fibrin ferment the influence of experiment (TGA).The figure is shown with single TFPI-106 (16 μ g/ml) and with rFVIIa (20 μ g/ml；2μg/ml；Or 0.2 μ g/ml) combination hemophilia A plasma it is solidifying Hemase generation figure (thrombogram).The hemophilia A plasma both without TFPI-106 or without rFVIIa is displayed that in figure And the fibrin ferment generation figure of non-haemophilic plasmas.
The fibrin ferment generation figure that Fig. 8 A describe is shown in the presence of 1pM tissue factors and 4 μM of phosphatide, in hemophilia A blood Exist in slurry with or without rFVIIa TFPI-106 or only exist the influence that rFVIIa generates fibrin ferment.Including non- Haemophilic plasmas is as control.
The fibrin ferment generation figure that Fig. 8 B describe is shown in the presence of the inhibitor of three Bethesda units (3BU), In the hemophilia A plasma of the platelet poor of Citrated exist with or without rFVIIa TFPI-106 or only exist The influence that rFVIIa generates fibrin ferment.Including non-haemophilic plasmas as control.Also include wherein adding TFPI-106 in figure The non-haemophilic plasmas control of (16 μ g/ml).
The fibrin ferment generation figure that Fig. 8 C describe is shown in the presence of the inhibitor of three Bethesda units (3BU), In the hemophilia B plasmas of the platelet poor of Citrated exist with or without rFVIIa TFPI-106 or only exist The influence that rFVIIa generates fibrin ferment.Including non-haemophilic plasmas as control.Also include wherein adding TFPI-106 in figure The non-haemophilic plasmas control of (16 μ g/ml).
Fig. 9 A are shown compared with the control, immediately to hemophilia A mouse administration of antibodies TFPI-106 (6mg/ after afterbody is cross-section ) and influences of the separated recombinant factor VIII (200 units/kg) to losing blood kg.Fig. 9 B are shown compared with control group, horizontal in afterbody Have no progeny 2 minutes to hemophilia A mouse administration of antibodies TFPI-106 (6mg/kg) and separated recombinant factor VIII (200 units/ Kg) the influence to losing blood.
Figure 10 A show that compared with recombinant factor VIII the TFPI 106 of various concentrations is to from heavy hemophilia A The influence in the clotting time of the whole blood of human patients.Figure 10 B are shown compared with recombinant factor VIII, the TFPI 106 of various concentrations The influence generated to the peak value fibrin ferment of the Platelet-rich plasm from the human patients with heavy hemophilia A.
Figure 11 A show the TFPI 106 of various concentrations to from the inhibitor with heavy hemophilia A and for FVIII The influence of the whole blood coagulation time of human patients.Figure 11 B show the TFPI 106 of various concentrations to from heavy hemophilia A And for FVIII inhibitor human patients Platelet-rich plasm peak value fibrin ferment generate influence.Figure 11 C are shown The TFPI 106 of various concentrations to from heavy hemophilia A and for FVIII inhibitor human patients blood platelet The influence in the clotting time of poor plasma.
Figure 12 A show that compared with recombinant factor VIII the TFPI 106 of various concentrations is to from osculant hemophilia A Human patients whole blood coagulation time influence.Figure 12 B are shown compared with recombinant factor VIII, the TFPI 106 of various concentrations The influence generated to the peak value fibrin ferment of the Platelet-rich plasm from the human patients with osculant hemophilia A.Figure 12 C Show blood coagulations of the TFPI106 of various concentrations to the platelet poor plasma from the human patients with osculant hemophilia A The influence of time.
Figure 13 A show that compared with recombinant factor IX the TFPI 106 of various concentrations is to from osculant hemophilia B The influence of the whole blood coagulation time of human patients.Figure 13 B show that compared with recombinant factor IX the TFPI 106 of various concentrations is to coming From the influence of the peak value fibrin ferment generation of the Platelet-rich plasm of the human patients with osculant hemophilia B.Figure 13 C are shown Clotting times of the TFPI 106 of various concentrations to the platelet poor plasma from the human patients with osculant hemophilia B Influence.
Figure 14 A show that compared with recombinant factor VIII the TFPI 106 of various concentrations from multidigit to having hemophilia A The influence of the whole blood coagulation time of human patients.Figure 14 B show that compared with recombinant factor VIII the TFPI 106 of various concentrations is right There is the influence of the peak value fibrin ferment generation of the Platelet-rich plasm of the human patients of hemophilia A from multidigit.Figure 14 C are shown The TFPI 106 of various concentrations is to having the clotting time of the platelet poor plasma of the human patients of hemophilia B from multidigit Influence.
Detailed description of the invention
1. general introduction
As noted above, haemophiliac has some hemostatic capabilities by its complete extrinsic pathway；But this is outer Source sexual approach is not enough to provide protection, because it is by tissue factor approach restrainer (TFPI) closing rapidly.Blocking/neutralize these trouble TFPI inhibitory action in person can compensate for FXa generations deficiency and standardize hemorrhagic diathesis.Therefore, the present invention discloses and example is special The opposite sex combines TFPI and suppresses its active antibody and its antigen-binding fragment.
2. definition
" activity for reducing TFPI " means that antibody or its antigen-binding fragment can：(i) for example, by the dilution based on blood plasma Prothrombin time measurement, compared with the setting time in the presence of without the antibody, the clotting time is reduced；(ii) example is passed through Such as Thrombectomy or rotation thrombus elasticity measure detection measurement, compared with the setting time in the presence of without the antibody, whole blood Clotting time is reduced；(iii) fibrin ferment generation increase；(iv) FXa activity is increased in the presence of TFPI；(v) in the presence of TFPI Strengthen platelet accumulation；(vi) fibrin generation is increased in the presence of TFPI；Or (vii) its any combinations.Antibody or antigen The inhibitory activity of binding fragment can, but must not be dose dependent (such as based on blood plasma dilution prothrombin time survey Measurement in fixed, causes clotting time dose dependent to reduce).
In addition, according to presently disclosed and exemplary, obtaining the Kunitz domains 2 of anti-TFPI antibody and TFPI, (K2 is tied Structure domain) eutectic structure.Structural analysis is shown and other disclosed TFPI antibody (it is used as refers to antibody in embodiment) phases Than exemplary antibodies of the invention identify the distinct epitopes of TFPI.It is and several for example, as shown in Figure 1A to 1F and Fig. 2A to 2E Compared with reference to TFPI antibody (R＆D (Mab2479) Fab, Novo2021 (also referred to as hz4F36) Fab, 2A8Fab), TFPI-23, Non-overlapped site in the K2 domains of TFPI-24 and 4D8 antibody bindings TFPI.
Therefore, in certain embodiments, antibody (and antigen-binding fragment) identification disclosed in this invention is located at TFPI K2 domains TFPI distinct epitopes.Alanine-scanning based on the eutectic structure and calculating, the epitope include following three A residue important to antibody-antigen interactions：Ile105, Arg107 and Leu131 are (according to SEQ ID NO：People shown in 2 The numbering of class TFPI).Antibody binding is caused to be lost into alanine this three residue mutations.For example, antibody TFPI-23 and its change Body (such as TFPI-106 and TFPI-107) identifies this epitope.
In certain embodiments, identify that critical epitopes residue disclosed by the invention allows the antibody (and its antigen binding Fragment) reduce TFPI activity.Especially, which shows that the K2 domains of TFPI use pyramidal structure, the cone Tip (especially Arg107) combines FXa.Both TFPI-23 and TFPI-24 identify the tip of this tapered zone and block TFPI with FXa is combined.Different epitopes in antibody 4D8 identification K2 domains.Although not direct interact with the residue at the cone tip, But 4D8 blocks TFPI to be combined with FXa.Table 15 is summarized when compared with other known TFPI antibody, by the exemplary of the present invention The non-overlapping epitopes residue of antibody identification.
In addition, in certain embodiments, antibody and its antigen-binding fragment disclosed by the invention have been shown for treating Clotting defect (such as hemophilia) and desired pharmacological activity and pharmacokinetic property for shortening the bleeding time.
Antibody with epitope " preferably in combination with " or " specific binding " (being used interchangeably in the present invention) is institute in this area Well known term, and measure the specificity or preferably in combination with method be also known in the art.Compared to the cell with replacement Or substance reaction or combination, if molecule with the compatibility of longer duration and/or bigger it is frequent, more quickly with it is specific Cell or substance reaction or combination, then the molecule be referred to as showing " specific binding " or " preferably in combination with ".Compared to The combination of other materials, if antibody with the compatibility (affinity) of bigger, affinity (avidity), be easier and/or with more The long duration is combined with target, then the antibody " specific binding " or " preferably in combination with " target.In addition, compared to it with being present in The combination of other materials in sample, if antibody with the compatibility of bigger, affinity, be easier and/or with it is longer lasting when Between combined with the target in sample, then the antibody " specific binding " or " preferably in combination with " target.For example, specific binding or preferred knot The antibody for closing TFPI epitopes be with for the combination compared with itself and other TFPI epitopes or non-TFPI epitopes with the compatibility of bigger, Affinity, the antibody for being easier and/or being combined with the longer duration epitope.By reading this definition it may also be appreciated that example Antibody (or part or epitope) such as " specific binding " or " preferably in combination with " the first target may not be specifically bound or excellent Choosing combines the second target.Therefore, " specific binding " or " preferably in combination with " are not necessarily required to (although it may include) single-minded combination.One As for, but not necessarily, refer to reference to mean preferably in combination with." specific binding " or " preferably in combination with " includes to identify and combining Specific molecular but the substantially compound of other molecules of nonrecognition or combination, such as protein, nucleic acid, antibody etc. in sample. For example, identify and combine the cognate ligand in sample (cognate ligand) or binding partners are (such as with reference to the anti-of TFPI TFPI antibody) but substantially nonrecognition or the antibody or peptide acceptor with reference to other molecules in sample, it is homologous to specifically bind this Ligand or binding partners.Therefore, under specified determination condition, the specific bound fraction (such as antibody or its antigen knot Close part or acceptor or its ligand binding moiety) it is present in test specimens preferably in combination with specific target molecule and not with significant quantity combination Other components in this.
Many measure form can be used to select the antibody or peptide that specifically bind molecule interested.Such as solid phase ELISA Immunoassays, immune precipitation, BiacoreTM(GE Healthcare, Piscataway, NJ), KinExA, the cell of fluorescent activation Sort (FACS), OctetTM(Fort é Bio, Inc, Menlo Park, CA) and western blot analysis are to can be used for identifying and resisting The antibody of former or acceptor or its ligand binding moiety (it specifically binds with cognate ligand or binding partners) specific reaction Measure many measure.In general, specificity or selective reaction more generally surpass two times of at least background signal or noise 10 times of background is crossed, more generally more than 50 times of background, more generally more than 100 times of background, more generally more than 500 times of background, Even more generally exceed 1000 times of background, and even more generally exceed 10000 times of background.In addition, work as equilibrium dissociation constant (KDDuring)≤7nM, which is referred to as " specifically binding " antigen.
In the present invention, term " binding affinity " is used as non-between two molecules (such as antibody or its fragment and antigen) The measurement of covalent interaction intensity.Term " binding affinity " is for describing unit price interaction (intrinsic activity).
Interact by unit price, can be by measuring dissociation constant (KD) quantify two molecules (such as antibody or its piece Section and antigen) between binding affinity.In turn, it can be used such as surface plasma body resonant vibration (SPR) method (Biacore) logical The formation of measurement compound and the dynamics dissociated are crossed to measure KD.It is normal corresponding to the speed of the association and dissociation of the unit price compound Number is referred to as association rate constant ka(or kon) and dissociation rate constants kd(or koff)。KDPass through equation KD=kd/kaAnd and ka And kdIt is associated.The numerical value of dissociation constant can directly be measured by known method, and even can be for example, by Caceci et al. (1984, Byte9：Method shown in 340-362) calculates composite mix.For example, KDMeasure can be used such as by Wong＆ Lohman(1993,Proc.Natl.Acad.Sci.USA 90:Double filter nitrocellulose filter knot disclosed in 5428-5432) Measure is closed to establish.The standard test of other binding abilities for being used to assess ligand (such as antibody for target antigen) is ability Known to domain, including, such as other place examples in ELISA, protein imprinted, RIA and flow cytometry, and the present invention Measure.The binding kinetics and binding affinity of antibody can also be assessed by standard test known in the art, such as surface Plasma resonance (SPR) (such as use BiacoreTMSystem or KinExA).
Being at war with property combination mensuration, wherein by another ligand of the combination of antibody and antigen and target and the target (such as Another antibody or the soluble recepter for being otherwise in connection with the target) combination compare.50% concentration suppressed occurs to be claimed For Ki.Under ideal conditions, the KiEqual to KD.The KiValue will never be less than KD, so KiMeasurement can be easily by being carried The K of confessionDThe upper limit substitutes.
According to above-mentioned definition, interacting with different molecular, (different antibodies are to referring to compared with such as relevant binding affinity Determine the binding affinity of antigen) can be by comparing the K of single antibody/antigenic compoundDValue compares.Antibody or other combinations are matched somebody with somebody The K of even bodyDValue this area can be used to improve the method established to measure.One kind is used to measure KDThe method of value is to use surface Plasma resonance (typically uses bio-sensor system, such asSystem) carry out.
Similarly, the specificity of interaction can be by measuring interaction interested (such as between antibody and antigen Specificity interaction) KDIt is worth and interacts (such as known control antibodies not combined with TFPI) with uninterested KDValue is assessed compared to relatively.
Specifically bind the antibody of its target (that is, can show low K as discussed above with high-affinityD) its target is combined, And other non-target molecules are combined with relatively low compatibility.Such as antibody can be with 1 × 10-6M or higher (preferably 1 × 10-5M or more Height, more preferably 1 × 10-4M or higher, more preferably 1 × 10-3M or higher, more preferably 1 × 10-2M or higher) KDValue combines non- Target molecule.Preferably, antibody of the invention can be higher by its at least 2 times of binding affinity with another non-TFPI molecules, 10 times, The compatibility of 50 times, 100 times, 200 times, 500 times, 1,000 times or 10,000 times or higher is combined with its target.
In general, TFPI antibody is needed with high-affinity combination TFPI to be effectively reduced the activity of TFPI.However, work as The binding affinity of the antibody is too high, which can soon be degraded by internalization (internalized) and by host cell. This can potentially result in short half-life period and duplicate injection.Such as antibody TFPI-23 shows that the combination low compared with TFPI-24 is affine Property (Kd), and in some cases, because it is with relatively low internalization speed and longer half-life period, it appears that be more advantageous to facing Bed uses.Therefore, binding affinity (Kd) is 5 × 10-7To about 5 × 10-11M, especially about 1 × 10-8To about 1 × 10-10M leads to It is often desired, the chronic disease (such as hemophilia) of duplicate injection is needed particularly for treatment.It without being limited to any specific Theory, which is considered effectively suppressing active required binding affinity of TFPI at (i) and (ii) is longer partly declines Balanced between phase and the antibody internalization of reduction.
Specific amino acid residue position in TFPI is according to SEQ ID NO：2 (mankind TFPI α K1K2K3) are numbered.So And the present invention is not limited to SEQ ID NO：2.Correspondence residue from other TFPI homologues, isotype, variation or fragment can Identified according to sequence alignment known in the art or structure alignment.Can be by craft or the known sequence of use for example, comparing Row alignment programs, such as ClustalW2 or " 2 sequences of BLAST ", are completed using default parameters.For example, SEQ ID NO：2 Arg107 corresponds to mouse TFPI K1K2 (SEQ ID NO：4) Arg104.
The ability that " antigen-binding fragment " of antibody refers to retain molecule of the antigen binding is (preferably with substantially the same Binding affinity) full length antibody fragment.The example of antigen-binding fragment includes (i) Fab fragments, by VL, VH, CL and CH1 The monovalent fragment of domain composition；(ii)F(ab')2Fragment, includes the Fab fragments two by the disulfide bond bridging of hinge area Bivalent fragment；(iii) the Fd fragments being made of VH and CH1 domains；(iv) it is made of VL the and VH domains of antibody single armed Fv fragments, dAb fragments (the Ward et al., (1989) Nature 341 that (v) is made of VH domains:544-546)；And (vi) separated complementary determining region (CDR), the Fv (dsFv) of disulfide bond connection and antiidiotype (anti-Id) antibody resist with intracellular Body (intrabody).In addition, although two domain VL and VH of Fv fragments by respectively gene code, using restructuring side Method, linking them by synthetic linker, (wherein the VL and VH areas are matched to form unit price so that they are made single protein chain Molecule (is known as scFv (scFv))；See, e.g. Bird et al.Science 242:423-426 (1988) and Huston et al.Proc.Natl.Acad.Sci.USA85:5879-5883(1988).Also the single-chain antibody of other forms is included, such as Including bivalent antibody (diabody).Bivalent antibody is divalence, bispecific antibody, and wherein VH and VL domains expression is single On polypeptide chain, but the connector used is so short that very much formation pairing between two domains that can not allow on same chain, so that Force the complementary domain of the domain and another chain to match and cause two antigen binding sites (see, e.g. Holliger et al.Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)；Poljak et al.,1994, Structure 2:1121-1123)。
Antibody " variable domains " refers to antibody light chain (VL) variable region or heavy chain of antibody variable region alone or in combination (VH).As it is known in the art, the variable region of heavy chain and light chain respectively passes freely through the four of three complementary determining region (CDR) connections A framework region (FR) composition, and participate in being formed the antigen binding site of antibody.
Residue in variable domains is numbered according to Kabat, and Kabat is the heavy-chain variable domains for the antibody that collects Or the numbering system of light variable domains.Referring to Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD.(1991).Using this numbering system, actual linear amino acid sequence can contain less or extra Amino acid, corresponding to the variable domains FR or CDR shorten or insertion.Such as heavy-chain variable domains may include H2's Single amino acid Insert Fragment (residue 52a (according to Kabat)) after residue 52 and it is inserted into after heavy chain FR residue 82 Residue (such as residue 82a, 82b and 82c (according to Kabat)).Specifying the Kabat numberings of the residue of antibody can have by comparing The antibody homologous region of " standard " Kabat numbered sequences determines.Various algorithms for distributing Kabat numberings are available.Unless It is otherwise noted, the present invention distributed the Kabat of variable region using Abysis (www.abysis.org) algorithm delivered in 2012 Numbering.
Specific amino acid residue position (such as paratope residue disclosed in this invention) in antibody also according to Kabat is numbered.
" complementary determining region " (CDR) can according to the accumulation both the definition of Kabat, Chothia, Kabat and Chothia, AbM, contact and/or conformation define or the method for any measure CDR known in the art is identified.See, e.g. Kabat Et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. (hypervariable region)； Chothia et al.,1989,Nature 342:877-883 (structure ring structure).The AbM definition of CDR be Kabat with The AbM antibody modeling softwares of compromise and use Oxford Molecular between ChothiaCDR's " connects Touch " define be based on observe antigen contact (be set forth in MacCallum et al., 1996, J.Mol.Biol., 262: In 732-745).It is (see, e.g. Makabe et based on the residue to antigen binding generation enthalpy contribution that " conformation " of CDR, which defines, al.,2008,Journal of Biological Chemistry,283:1156-1166).Also other CDR boundary definitions can Can not follow strictly one of above method, but will with Kabat CDR at least a portion it is overlapping, it is but in view of pre- Survey or experiment find that specific residue or residue group or even whole CDR are not significantly affected by antigen binding, they may shorten or Extend.As used in the present invention, CDR can refer to the CDR defined by any method (combination for including method) as known in the art.
In embodiment (referring to table 3 and 4), which is defined as follows (numbers according to Kabat；H：Heavy chain；L：Light chain)：
CDR-H1:H26-H35B；CDR-H2:H50-H65；CDR-H3:H95-H102
CDR-L1:L24-L34；CDR-L2:L50-L56；CDR-L3:L89-L97
" framework " (FR) residue is the constant region for immunoglobulin sequence residue in addition to CDR residues.VH or VL domain framework bags Containing four framework sub-districts：FR1, FR2, FR3 and FR4, are interspersed with the CDR with lower structure therebetween：FR1-CDR1-FR2-CDR2- FR3-CDR3-FR4.In embodiment (referring to table 3 and 4), FR residues include following (numbered according to Kabat；H：Heavy chain；L：Gently Chain)：
" epitope " refers to antigen (Ag) area or region that antibody is specifically bound, such as comprising mutual with the antibody (Ab) The area or region of the residue of effect.Epitope can be linear or conformation.In linear epitope, protein and interacting molecule (example Such as antibody) between the points of all interactions linearly exist along the primary amino acid sequence of protein." non-linear epitope " Or " comformational epitope " includes the noncontinuity polypeptide (or amino acid) in antigen protein, the antibody of the epitope is specific to With reference to the antigen protein.Term " epitope " used in the present invention is defined as by any method known in the art A part for the antigen that can be combined with antibody specificity of (such as being measured by routine immunization) measure.Alternatively, in discovery procedure In, the generation of antibody and characterization can illustrate the information related with desired epitope.Pass through the information, the knot of contestable screening afterwards Close the antibody of same epitope.The method for reaching this purpose is to be at war with to study with cross competition to contend with one other or intersect to find out The antibody of competition binding TFPI.That is, antibody competition combination antigen, so that the antibody competition combination disclosure is anti- The antigen binding site of TFPI antibody.
Term " paratope " is by reversing the viewpoint of the definition of above-mentioned " epitope " to derive, and refers to participate in anti- The area or region for the antibody molecule that original combines, such as area or region comprising the residue with antigen interactions.Paratope It is but linear or conformation (such as discontinuous residue in CDR).
Epitope/the paratope for the antibody/antigen combination pair specified can utilize the epitope mappings of various experiments and calculating Method is defined and characterized with different level of detail.Experimental method includes mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectrum Learn, the method that hydrogen/deuterium exchanges mass spectrum (HX-MS) and various competition bindings.Since each side's genealogy of law is dependent on unique principle, epitope Description with measure its method tight union.It is therefore intended that epitope/paratope of antibody/antigen pair will be according to institute The drawing method of use and have different definition.
In most detailed level, epitope/paratope for interacting between antibody and antibody can pass through restriction It is present in the space coordinate of the atomic contacts in Ag-Ab interactions and on their letters to the thermodynamic (al) Relative Contribution of combination Cease to define.In a kind of level, epitope/paratope residue is by limiting the space coordinates of the atomic contacts between Ag and Ab To characterize.On the one hand, epitope/paratope residue can be defined by specific criteria, for example, the atom in Ab and Ag it Between distance (such as the distance of the heavy atom of the heavy atom of homologous antibody and antigen is equal to or less than(" contact " residue)). On the other hand, epitope/paratope residue is characterized in that participating in the interaction of hydrogen bond and homologous antibody/antigen, or Interaction with hydrone so as to the hydrone also also with the homologous antibody/antigen Hydrogenbond (hydrogen bond knot of water mediation Close).On the other hand, epitope/paratope residue is characterized in that forming salt bridge with the residue of the homologous antibody/antigen. On the other hand, being characterized by with the homologous antibody/antigen interactions for epitope/paratope residue and hiding Surface region (BSA) in have non-zero change residue.Under less detailed level, epitope/paratope can pass through Function (such as with other Ab competition bindings) characterizes.Epitope/the paratope also can by more upper be defined as including ammonia Base acid residue, will change the interaction characteristic between the Ab and Ag when the amino acid residue is by another 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as Alanine-scanning).
Limited in the space coordinate of the compound by antibody (such as Fab fragments or two Fab fragments) between its antigen Under the background of crystal structure derived from fixed X-ray, unless otherwise indicated, epitope residues refer to TFPI residues as described below (i) there is heavy atom (that is, non-hydrogen atom), the distance of itself and the heavy atom of homologous antibody existsWithin (also referred to as " contact " residual Base), (ii) participates in the residue Hydrogenbond with homologous antibody, or is combined with water molecules hydrogen bond, so that this hydrone is also same with this Source antibody Hydrogenbond (Hydrogenbond mediated by water), (III) participates in the salt bridge of the residue of the homologous antibody, and/or (iv) by In with homologous antibody interaction and with non-zero change in hiding surface region (BSA).In general, BSA is applied Add and block, to avoid comprising with the residue at least to interact.Therefore, unless otherwise indicated, if the BSA of the epitope residues isOr the epitope residues of the electrostatic interaction, then selection classification (iv) when bigger or participation antibody binding TFPI.Similarly, Unless otherwise indicated or contradicted by context, under the background of crystal structure derived from X-ray, paratope residue refers to Antibody residue (i) as described below has heavy atom (that is, non-hydrogen atom), and the distance of itself and the heavy atom of TFPI existsWithin (also referred to as " contacting " residue), (ii) participates in being combined with TFPI residues Hydrogenbond or with water molecules hydrogen bond, so that this hydrone Also participate in forming salt bridge, and/or (iv) with the residue of TFPI with TFPI Hydrogenbonds (Hydrogenbond mediated by water), (iii) There is non-zero change in hiding surface region (BSA) due to interacting with TFPI.Furthermore unless otherwise indicated, if The BSA of the paratope residue isOr electrostatic interaction when bigger or participation antibody binding TFPI, then select class The not paratope residue of (iv).
According to used epitope mapping procedure and with the description as described in epitope and definition of the acquisition of different level of detail The fact, it follows the ratio work of the epitope of the more different Ab of epitope on identical Ag can be under different detailed levels with similar Method carries out.Such as if it contains identical amino acid residue group, with the epitope of amino acid hierarchy description (such as from X-ray Structure determination) be referred to as it is identical.If the epitope does not share amino acid residue, it is independent that these epitopes, which are recognized, (unique).And if the combination of corresponding antibody is that mutual exclusion (that is, can exclude concurrently or consecutively anti-with other with an antibody binding Body combination), then it is overlapping to be characterized in that the epitope of competition binding is referred to as having；And if the antigen can accommodate at the same time it is corresponding with two kinds Antibody binding, then the epitope be referred to as independent (unique).
The epitope and paratope for the antibody/antigen pair specified can be identified by conventional method.For example, epitope is substantially Position can by assess antibody binding different fragments or variation TFPI polypeptides ability measure (this above elsewhere have it is more complete Description).Conventional method (such as institute in embodiment can also be used in the specific residue contacted in TFPI with the specific residue in antibody Description) measure.For example, antibody/antigen compound crystallizableization.The crystal structure can through measure and for differentiate antibody with The specific site to interact between antigen.
The term " competition " on antibody means first antibody or its antigen-binding portion thereof with resisting as used in the present invention Former combination reduces the combination of subsequent secondary antibody or its antigen-binding portion thereof and the same antigen.In general, with reference to first Antibody can create steric hindrance, conformation change or be bound to common epitope (or part thereof) so that secondary antibody and the phase The combination of synantigen is reduced.Standard Competition measure can be used for whether two kinds of antibody of measure contend with one other.One kind is suitable for antibody The measure of competition is directed to use with Biacore technologies, which can utilize surface plasma body resonant vibration (SPR) technology (logical Often using bio-sensor system (such asSystem)) measure the degree to interact.For example, SPR can be used for External competitive binding suppresses measure to measure the ability that a kind of antibody suppresses secondary antibody and combines.It is another competing for measuring antibody The measure striven is to use the method based on ELISA.In addition, one kind is based on described in International Patent Application No. WO2003/48731 The high throughput method for " branch mailbox " antibody of the competition of antibody.If a kind of antibody (or fragment) reduces another antibody (or piece Section) with the combination of TFPI, then exist and compete.For example, continuously competition assay can be combined by sequentially adding different antibodies use.Add Enter first antibody and be likely to be breached combination close to saturation.Then, secondary antibody is added.If it is not detected by secondary antibody to tie with TFPI Close, or compared with the parallel determination (its numerical value may be set to 100%) in the presence of no first antibody, the knot of secondary antibody and TFPI Conjunction substantially reduce (such as reduce at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, At least about 60%, at least about 70%, at least about 80% or at least about 90%), then two kinds of antibody are considered as to compete with one another for.Implement The exemplary antibodies competition assay (and overlapping epitope analysis) carried out by SPR is provided in example 6.
The anti-TFPI antibody of the disclosure can have to be retouched with another antibody competition or cross competition of the disclosure and the present invention The ability that the TFPI stated is combined.For example, the antibody of the disclosure can with antibody competition or cross competition described in the invention with TFPI is combined or combined with by the suitable TFPI fragments or variation of antibody binding disclosed by the invention.
Combined that is, if the first anti-TFPI antibody is competed with secondary antibody with TFPI, but when secondary antibody is tied first It is not competed during conjunction TFPI, then this first antibody is considered as and secondary antibody " competition " (also referred to as unidirectional competition).Work as primary antibody Body and another antibody competition, no matter which kind of antibody is combined with TFPI first, then the antibody and other antibody " cross competition " and TFPI is combined.It is this competition or cross-competing antibodies can based on its in standard combination mensuration its with the present invention known antibodies it is competing Strive/the ability of cross competition identification.For example, SPR can be used (such as by using BiacoreTMSystem), ELISA measure or stream Formula cell art proves competition/cross competition.This kind of competition/cross competition may imply both antibody bindings to identical, again Folded or similar epitope.
Therefore, the anti-TFPI antibody of the disclosure can be identified by the method comprising combination mensuration, and combination mensuration assessment is surveyed Try antibody whether can with the reference antibody of the disclosure (such as TFPI-3, TFPI-21, TFPI-23, TFPI-24, TFPI-26, TFPI-106、TFPI-107、TFPI-108、TFPI-109、TFPI-110、TFPI-111、TFPI-112、TFPI-113、TFPI- 114th, 4D8,6B7.c5,7A4.D9) competition/cross competition combine the target molecule on binding site.
" Fc fusions " albumen is that wherein one or more polypeptides are the protein of Fc polypeptides of being operably connected.Fc fusion proteins Merge the Fc areas of immunoglobulin with fusion partner.
The binding affinity of antibody can represent that it refers to the dissociation rate of specific antigen-antibody interaction with Kd values.Kd It is the speed (also referred to as " dissociation rate (off-rate) (Koff) ") of dissociation to the speed (or " on-rate (kon) ") of combination Ratio.Therefore, Kd values are equal to Koff/kon and are represented with molar concentration (M), and Kd values are smaller, and binding affinity is stronger.It is anti- The method measure improved and established in this area can be used in the Kd values of body.A kind of illustrative methods for being used to measure Kd be surface etc. from Daughter is resonated (SPR), typically uses bio-sensor system, such asSystem.BIAcore dynamic analyses Comprising analysis antigen with its surface with fixed member (such as the molecule for including epitope binding structural domain) chip combined and Dissociation.The method of another Kd for measuring antibody is to use Bio-Layer interferometries, is typically used Technology (Octet QKe systems, ForteBio).Alternatively or additionally, it is possible to use can be from Sapidyne Instruments (Boise, Id) is obtained(dynamics excludes measure) measure.
Term " therapeutically effective amount " means to sufficiently achieve expected purpose (such as increase blood coagulation or in the case of hemophilia Shorten setting time or otherwise cause measurable benefit in subject in need) anti-TFPI antibody or its piece Section or the amount of combination comprising the antibody or its fragment.Accurate amount will depend on many factors, and including but not limited to this is controlled Component and physical characteristic, the consideration etc. of predetermined patient population, few patients of composition are treated, and can be by those skilled in the art Determine.
Term " synergistic treatment effective dose " means to work as to be provided together with second therapeutic agent (such as factor VIIa (FVIIa)) When, there is provided measurable benefit (such as reduce the clotting time, reduce the bleeding time, increase fibrin generation, enhancing blood platelet Accumulation etc.) anti-TFPI antibody higher than the cumulative measurable benefit of each therapeutic agent or antibody being administered alone or its antigen binding The amount of fragment.
Term " treatment " includes preventative and/or therapeutic treatment.If the treatment is applied before the clinical manifestation of illness With then it is considered preventative.Therapeutic treatment includes, such as improves or reduce the seriousness of disease or shorten disease Length.
If terminology used in the present invention is " about " +/- the 10% of exponential quantity.
3. anti-TFPI antibody
It is presently disclosed and it is exemplary be bind tissue factor approach restrainer (TFPI) antibody (and its antigen binding Fragment).The distinct epitopes of the antibody and antibody fragment combination TFPI.In certain embodiments, some tables in TFPI are identified Position residue can make the activity of the antibody (and its antigen-binding fragment) reduction TFPI.In addition, in certain embodiments, the present invention Disclosed antibody (and its antigen-binding fragment) is proved for treating clotting defect and reducing the desired medicine in bleeding time Learn activity and pharmacokinetic property.
A. tissue factor approach restrainer (TFPI)
TFPI is the multivalence Kunitz domains containing protease inhibitors.The mankind, mouse, machin, rabbit are provided in table 2 The exemplary sequence of son and rat TFPI.
Mankind TFPI is extracellular glycoprotein, it has two kinds of principal modes, TFPI- α and TFPI- β.(it is that have to TFPI α The glycosylation albumen (MW 43kD) of 276 amino acid) it is the largest TFPI forms and by three Kunitz spline structures domains and substantially C-terminus district's groups into.Variable sheer produces TFPI- β, it includes Kunitz domains 1 (K1) and Kunitz domains 2 (K2), but C-terminal part containing the replacement for lacking Kunitz domains 3 (K3) and basic region.TFPI- β are with glycosyl-phosphatidyl inositol (GPI) anchorin anchors to cell membrane by posttranslational modification.
The main target of TFPI is protease Factor Xa (FXa) and factor VIIa (FVIIa), these are coagulation cascade starting ranks Key factor in section.Biochemical analysis has disclosed the inhibitor that K2 is FXa, and K1 suppression FVIIa- tissue factors are compound Thing.The effect of K3 is unclear, because it seems do not have direct protease inhibiting activity, but can be as co-factor Protein S Recognition site.C-terminal domain may participate in the identification of prothrombinase on platelet surface specific to TFPI- α.
Kunitz domains 1 (K1) correspond to SEQ ID NO：2 amino acid residue 26-76, and Kunitz domains 2 (K2) SEQ ID NO are corresponded to：2 residue 91 to 147.K1 from other TFPI homologues, isotype, variation or fragment and K2 domains can be by for SEQ ID NO：2 sequence alignment or structure alignment are identified.
The TFPI of the disclosure includes any naturally occurring form of TFPI, these existing native forms, which can be derived from, appoints What suitable organism.For example, TFPI can be mammal TFPI, for example, the mankind, mouse, rat, non-human primates, ox, Sheep, dog, cat or pig TFPI.In certain embodiments, TFPI is mankind TFPI.TFPI can be the TFPI of mature form (i.e., The TFPI albumen of post translational processing is undergone in suitable cell).Such as this mature T FPI albumen can be glycosylated.
The TFPI of the disclosure includes any functional fragment or variation derived from naturally occurring TFPI.The function of TFPI Property fragment can be retain TFPI activity (such as inhibiting factor Xa (FXa), suppress FVIIa- tissue factor complexes activity And/or the ability as blood coagulation or the negative regulation agent of hemostasis) any TFPI composition parts or part.For example, functional fragment Kunitz domains can be included, such as both the K1 domains of TFPI, K2 domains or K1 and K2 domains.
Compared with naturally occurring TFPI, functional variant thereof can include one or more mutation and still retain naturally occurring The activity of TFPI, such as the ability of inhibiting factor Xa (FXa) or the active ability for suppressing FVIIa- tissue factor complexes.Example Such as, variation and SEQ ID NO：1st, 2,3,4,5,6 or 7 can have a different degrees of sequence thereto, for example, with SEQ ID NO： 1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6 or SEQ ID NO：In 7 Listed sequence at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% is identical.
TPFI fragments, variation, isotype and the homologue of the present invention should keep important epitope residues (if such as using TFPI-23 and TFPI-24 antibody, then be Ile105, Arg107 and Leu131).In addition, TFPI can include 5 or more, 8 or More, 10 or more, the surfaces of 12 or more or 15 or more the K2 domains of a TFPI can and residue.Surface Can and residue be with higher than 40% opposite accessibility residue.
For example, for TFPI K2 domains (see, e.g. SEQ ID NO：2), following amino acid residue has and is higher than 40% opposite accessibility：94-95,98,100-110,118-121,123-124,131,134,138-142 and 144-145. TFPI can include 5 or more, 8 or more, 10 or more, 12 or more or 15 or more these residues, such as TFPI fragments including 5 or more, 8 or more, 10 or more, 12 or more or 15 or more these residues.
B. anti-TFPI antibody
The K2 domains of the antibody of the present invention or its antigen-binding fragment specific binding TFPI can simultaneously suppress itself and FXa's Interaction and/or the activity for reducing TFPI.
FPI-23 and variation
On the one hand, the present invention includes preparing to increase the antibody of human framework's system genitale residue (" system genitale ") content The variation of TFPI-23 and TFPI-23.For example, TFPI-106 includes the mutation (Kabat numberings) of H1Q to E and H5V to L, and TFPI-107 includes the mutation (Kabat numberings) H1Q to E, H5V to L and H94I to K.For the purpose of the present invention, TFPI-23 Parental antibody and TFPI-106 system genitales variation can exchange in its epitope residues and the interaction of paratope residue.
On the one hand, the present invention provides the Kunitz domains 2 of specific binding tissue factor approach restrainer (TFPI) (K2) the separated antibody or its antigen-binding fragment of the epitope in, wherein the epitope include residue Ile105, Arg107 and Leu131 is (according to SEQ ID NO：2 numbering).In certain embodiments, the antibody or its antigen-binding fragment do not combine The Kunitz domains 1 (K1) of TFPI.
It is as disclosed in the present invention and exemplary, the K2 structures of TFPI are had found based on eutectic structure and calculating Alanine-scanning Distinct epitopes in domain.Especially, which shows that the K2 domains of TFPI use pyramidal structure, the tip of the cone (especially Arg107) combines FXa.TFPI-23, TFPI-24 and its variation identify residue near the tip of this tapered zone simultaneously TFPI is blocked to be combined with FXa.Therefore, epitope residues of the identification near the cone tip especially have for suppressing TFPI activity With.
In certain embodiments, the invention discloses include three residues important to antibody-antigen interactions TFPI epitopes：Ile105, Arg107 and Leu131 are (according to the numbering of mankind TFPI, such as SEQ ID NO：Shown in 2).By this three Residue mutations cause TFPI-23, TFPI-24 and its variation to combine and lose into alanine.Referring to table 28, it summarizes Alanine-scanning As a result.
Also extra TPFI residues are identified and participate in antibody binding, but these residues can be mutated into alanine without aobvious Write unstable effect.Referring to table 28.Therefore, in certain embodiments, which further includes one or more and is selected from With the residue of the following group：Cys106, Gly108, Cys130, Gly132 are (according to SEQ ID NO：2 numberings) and its any combinations.This A little epitope residues can be identified by TFPI-23, TFPI-24 and its variation.Referring to table 27, it shows TFPI-23 and TFPI-24 institutes Shared common epitope residues.
In certain embodiments, which further includes one or more and is selected from the residue of the following group：Asp102、 Arg112, Tyr127, Gly129, Met134, Glu138 are (according to SEQ ID NO：2 numberings) and its any combinations.These epitopes Residue can be identified by TFPI-23 and its variation (such as TFPI-106, TFPI-107), but not known by TFPI-24 (and its variation) Not.Referring to table 27.
In certain embodiments, which does not include one or more and is selected from the residue of the following group：E100、E101、P103、 Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, L140 are (according to SEQ ID NO：2 numberings) and its any combinations.Referring to table 27.According to WO201007269 (Novo Nordisk Co., Ltd), reference antibody 4F36 identifications Include following epitope：E100、E101、P103、Y109、T111、Y113、F114、N116、Q118、Q121、C122、E123、 R124, F125, K126 and L140.
In certain embodiments, which does not include one or more and is selected from the residue of the following group：D31、D32、P34、 C35, K36, E100, E101, P103, Y109, K126, G128 are (according to SEQ ID NO：2 numberings) and its any combinations.Referring to table 27.According to table 27, reference antibody 2A8 and 2A8-200 identification include following epitope：D31、D32、P34、C35、K36、E100、 E101, P103, Y109, K126 and G128.
In certain embodiments, which, which can refer to one or more TFPI " contact " residues, (has apart from homologous antibody Heavy atomInterior heavy atom (that is, non-hydrogen atom)), and be selected from comprising one or more with the residue of the following group：Asp102、 Gly104、Ile105、Cys106、Arg107、Gly108、Arg112、Tyr127、Gly129、Cys130、Leu131、Gly132、 Met134, Glu138 are (according to SEQ ID NO：2 numberings) and its any combinations.Referring to table 29B.
In certain embodiments, the epitope can refer to one or more participate in antibody residue or with hydrone (its also with together Source antibody Hydrogenbond, water mediation Hydrogenbond) Hydrogenbond TFPI residues, and comprising one or more be selected from the following group Residue：Asp102, Arg107, Arg 112, Tyr127 and Leu131 are (according to SEQ ID NO：2 numberings) and its any combinations.This A little epitope residues participate in and homologous antibody Hydrogenbond.Referring to table 29B.
In certain embodiments, which can refer to due to interacting with homologous antibody and in hiding surface region (BSA) residue of non-zero change is included in, and is selected from comprising one or more with the residue of the following group：Asp102、Gly104、 Ile105、Cys106、Arg107、Gly108、Arg112、Tyr127、Gly129、Cys130、Leu131、Gly132、Asn133、 Met134, Glu138 are (according to SEQ ID NO：2 numberings) and its any combinations.These are referring to table 29B.
Any combinations of these different classes of epitope residues are also contained in the present invention.
In certain embodiments, the epitope include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, At least 8, at least 9, at least 10, at least 11, at least 12, at least 13 or all above-mentioned epitope residues or above-mentioned various types of other table Any combinations of position residue.
The paratope residue of TFPI-23 (and variation) can refer to following contact residues (positioned at TFPI epitope residuesIt is interior)：H33Ala、H47Trp、H50Ala、H51Ile、H52Ser、H56Ser、H58Tyr、H95Leu、H96Gly、H97Ala、 H98Thr, H99Ser, H100Leu, H100A Ser, L29Ala, L31Tyr, L91Tyr, L95A Ser, L95B Gly and L95C Ser；And optionally, L93Ser and L96Gly (being numbered according to Kabat).L93SerAnd L96GlyIt is to appoint Choosing because the distance only slight beyondIt is but close enough to being rounded to
It is noted that above-mentioned contact residues are the Original Residues from TFPI-23 antibody.However, it is based on structural analysis And Alanine-scanning, it is believed that many contact residues in TFPI-23 can be substituted without significantly affecting antigen knot by another residue Close.For example, table 29A shows that many contact residues in TFPI-23 can be substituted by other residues and only combination or stability are made Into<0.5kcal/mol influence ("<0.5kcal/mol " means that substitution has neutral effect to combining).Especially, such as table 29A Shown in 4 row, 3 CDR positions and 1 framework positions：H47, H58, L91 and L96 (being numbered according to Kabat) are only capable of allowing one or two A residue：(a) H47 is Trp or Tyr；(b) H58 is Tyr；(c) L91 is Tyr or Arg；And (d) L96 is Gly or Asn.Such as table What 29A the 4th was concluded in arranging, other CDR positions can accommodate more polysubstituted.
Therefore, in certain embodiments, antibody described in the invention or its antigen-binding fragment include following residue (being numbered according to Kabat)：
(a) H33 is Ala, Asn, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val；
(b) H47 is Trp or Tyr；
(c) H50 is Ala, Arg, Gly, Lys, Met, Phe, Pro, Ser, Thr, Tyr or Val；
(d) H51 be Ile, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；
(e) H52 be Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val；
(f) H56 is Ser, Arg, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；
(g) H58 is Tyr；
(h) H95 is Leu, Gln, Ile, Phe or Tyr；
(i) H96 is Gly, Ala, Arg, Asn, Asp, Gln, Ile, Lys, Met, Phe, Pro, Ser, Thr or Val；
(j) H97 be Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；
(k) H98 be Thr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；
(1) H99 is Ser, Ala, Gly, Phe or Pro；
(m) H100 is Leu, Arg, His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr or Val；
(n) H100A is Ser, Ala, Arg, Asn, Asp, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Ser, Thr Or Trp；
(o) L29 be Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；
(p) L31 be Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；
(q) L91 is Tyr or Arg；
(r) L95A be Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；
(s) L95B be Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；And
(t) L95C be Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；
And optionally comprising (u) L93 be Tyr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val；And
(v) L96 is Gly or Asn.
In these residues, H47 is Framework residues；It is CDR residues that other are all.
When applying tightened up substitution standard-substitution must lead to<The compatibility of -0.5kcal/mol, it means that take In generation, must be following (being numbered according to Kabat) (the 5th columns of table 29A) with actively (neutral/to stablize) effect-contact residues：
(a) H33 is Ala or Val；
(b) H47 is Trp；
(c) H50 is Ala；
(d) H51 is Ile；
(e) H52 is Ser, Arg, Lys, Phe or Tyr；
(f) H56 is Ser, Arg or Lys；
(g) H58 is Tyr；
(h) H95 is Leu；
(i) H96 is Gly, Ala, Arg, Asn, Lys, Pro, Ser or Val；
(j) H97 is Ala；
(k) H98 is Thr, His, Ile, Leu, Met, Phe or Tyr；
(1) H99 is Ser；
(m) H100 is Leu, Phe, Trp or Tyr；
(n) H100A is Ser, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, Phe, Pro or Trp；
(o) L29 is Ala；
(p) L31 is Tyr；
(q) L91 is Tyr；
(r) L95A is Ser, Phe, Trp or Tyr；
(s) L95B is Gly；And
(t) L95C is Ser, Arg, Asn, Gln, Glu, Ile, Leu, Lys, Met, Phe, Trp, Tyr or Val；
And optionally：(u) L93 is Ser；And
(v) L96 is Gly.
Additionally or alternatively, the 6th row that acceptable substituted selection can be based on table 29A, wherein preceding 3 residues are right according to its The influence of compatibility is selected (that is, to preceding 3 predictions site of compatibility most stabilization).According to this standard, contact residues (numbered as follows according to Kabat)：
(a) H33 is Ala, Val, His or Phe；
(b) H47 is Trp or Tyr；
(c) H50 is Ala, Thr, Ser or Phe；
(d) H51 is Ile, Arg, Lys or Pro；
(e) H52 is Ser, Phe, Arg or Tyr；
(f) H56 is Ser, Lys, Tyr or Phe；
(g) H58 is Tyr；
(h) H95 is Leu, Ile, Gln or Phe；
(i) H96 is Gly, Arg, Asn or Lys；
(j) H97 is Ala, Leu, Tyr or Ile；
(k) H98 is Thr, Tyr, Phe or His；
(1) H99 is Ser, Pro, Ala or Phe；
(m) H100 is Leu, Tyr, Trp or Phe；
(n) H100A is Ser, Arg, Leu or Trp；
(o) L29 is Ala, Glu, Asp or Gln；
(p) L31 is Tyr, Glu, Asp or Trp；
(q) L91 is Ty or Arg；
(r) L95A is Ser, Phe, Tyr or His；
(s) L95B is Gly, Glu, Asp or Pro；And
(t) L95C is Ser, Trp, Tyr or Phe；
And optionally (u) L93 is Ser, Glu, Asp or His；
(v) L96 is Gly or Asn.
The paratope residue of TFPI-23 (and variation) also can refer to participate in the residue of TFPI or with hydrone (its With TFPI Hydrogenbonds) residue of Hydrogenbond, and comprising following：H58Tyr、H96 Gly、H97Ala、H98Thr、H99Ser、 H100Leu, L29Ala, L31Tyr and L95B Gly (are numbered) according to Kabat.Referring to table 29B.
The paratope residue of TFPI-23 (and variation) also can refer in BSA have due to interacting with TFPI The residue of non-zero change, and comprising following：(being numbered according to Kabat)：H33Ala、H58Tyr、H95Leu、H96Gly、H97Ala、 H98Thr, H99Ser, H100Leu, H100A Ser, L29Ala, L31Tyr, L91Tyr, L93Ser, L95A Ser and L95B Gly.Using blocking, (BSA isOr bigger, or participate in electrostatic interaction) to avoid comprising with minimum interaction Residue.Referring to table 29B.
If not applying blocking for BSA, paratope residue includes following：H33Ala、H34Met、H47Trp、H50Ala、 H51Ile、H52Ser、H56Ser、H58Tyr；H95Leu、H96Gly、H97Ala、H98Thr、H99Ser、H100Leu、H100A Ser, L28Gly, L29Ala, L31Tyr, L91Tyr, L93Ser, L94Ser, L95A Ser, L95B Gly, L95C Ser and L96Gly.Referring to table 29C.
The antibody or its antigen-binding fragment of the present invention can be identical with reference to being combined with the antibody of concrete example of the present invention TFPI epitopes or domain.For example, antibody or its antigen-binding fragment can be by comparing combination and the TFPI-23 of itself and TFPI Or system genitale variation (such as TFPI-106 and TFPI-107) and the combination of TFPI are identified；Or by compare these antibody with The function of TFPI-23 and its variation is identified.Analysis and measure available for these discriminating purposes include assessing competition binding The measure of TFPI will simultaneously illustrate in embodiment.
In one embodiment, antibody of the invention or antigen-binding fragment can combine and antibody described in the invention The identical epitope or region that (such as TFPI-23 and its variation) is combined.This may include itself and specific TFPI as described above Contact residues.For example, the present invention antibody or antigen-binding fragment can with selected from the following group contact residues (It is interior) Mode is combined with TFPI：Asp102、Gly104、Ile105、Cys106、Arg107、Gly108、Arg112、Tyr127、 Gly129, Cys130, Leu131, Gly132, Met134, Glu138 are (according to SEQ ID NO：2 numberings) and its any combinations.This The antibody of invention or its antigen-binding fragment can have the ability to be combined with the epitope of the residue of the following group with being selected from comprising one or more： Asp102、Gly104、Ile105、Cys106、Arg107、Gly108、Arg112、Tyr127、Gly129、Cys130、Leu131、 Gly132, Met134, Glu138 are (according to SEQ ID NO：2 numberings) and its any combinations.
Antibody and its antigen-binding fragment can include it is at least one on TFPI at least one epitope residues (according to SEQ ID NO：2 numberings)Interior paratope residue (being numbered according to Kabat), it is as follows：Epitope residues 102Asp is located at paratope residue H58It is interior；Epitope residues 104Gly is located at paratope residue H58It is interior；Epitope residues 105Ile be located at paratope residue H33Ala, H50Ala, H51Ile, H52Ser, H56Ser、H58Tyr、H95It is interior；Epitope residues 106Cys is located at paratope residue H100Leu and H100AIt is interior；Epitope residues 107Arg is located at paratope residue H96Gly, H97Ala, H98Thr, H99Ser, H100It is interior；Epitope residues 108Gly is located at paratope residue H100It is interior；Epitope residues 112Arg is located at Paratope residue L29Ala, L31It is interior；Epitope residues 127Tyr is located at paratope residue L31It is interior；Epitope residues 129Gly is located at paratope residue L31It is interior；Epitope residues 130Cys is positioned at anti- Former paratope residue L91Tyr, L95BIt is interior；Epitope residues 131Leu be located at paratope residue H47Trp, H50Ala、H58Tyr、L95A Ser、L95B Gly、L95CIt is interior；It is residual that epitope residues 132Gly is located at paratope Base H58Tyr, L95A Ser, H58Tyr, L95AIt is interior；Epitope residues 134Met is located at paratope residue L95AIt is interior；And epitope residues 138Glu is located at paratope residue L29It is interior.Referring to table 29A and 29B.
Antibody or its antigen-binding fragment can also include it is at least one can be with the epitope residues of TFIP (according to SEQ ID NO： 2 numbering) formed hydrogen bond paratope residue (being numbered according to Kabat), it is as follows：Epitope residues 102Asp can be with antigen Paratope residue H58Tyr forms hydrogen bond；Epitope residues 107Arg can be selected from the paratope residue of the following group with least one Form hydrogen bond：H96Gly, H97Ala, H98Thr, H99Ser and H100Leu；Epitope residues 112Arg can be residual with paratope Base L29Ala forms hydrogen bond；Epitope residues 127Tyr can form hydrogen bond with paratope residue L31Tyr；And epitope residues 131Leu can form hydrogen bond with paratope residue L95B Gly.Referring to table 29B.
Antibody or its antigen-binding fragment can also include it is at least one due to epitope residues (according to SEQ ID NO：2 compile Number) interact and there is the paratope residue (being numbered according to Kabat) of non-zero change in BSA, it is as follows：Epitope is residual Base 102Asp and paratope residue H58Tyr interacts；Epitope residues 104Gly and paratope residue H58Tyr phases Interaction；Epitope residues 105Ile is interacted with least one be selected from the paratope residue of the following group：H33Ala、 H34Met, H50Ala, H51Ile, H52Ser, H56Ser, H58Tyr and H95Leu；Epitope residues 106Cys and at least one choosing From the paratope residue interaction with the following group：H95Leu, H100Leu, H100A Ser and L91Tyr；Epitope residues 107Arg is interacted with least one be selected from the paratope residue of the following group：H96Gly、H97Ala、H98Thr、 H99Ser and H100Leu；Epitope residues 108Gly and paratope residue H100Leu interacts；Epitope residues 112Arg Interacted with least one be selected from the paratope residue of the following group：L29Ala, L31Tyr and L93Ser；Epitope residues 127Tyr is interacted with least one be selected from the paratope residue of the following group：L31Tyr and L95B Gly；Epitope residues 129Gly is interacted with least one be selected from the paratope residue of the following group：H100A Ser, L31Tyr and L91Tyr； Epitope residues 130Cys is interacted with least one be selected from the paratope residue of the following group：H95Leu、H100A Ser、 L31Tyr, L91Tyr and L95B Gly；Epitope residues 131Leu is selected from the paratope residue phase of the following group with least one Interaction：H47Trp, H50Ala, H58Tyr, H95Leu, L31Tyr, L91Tyr, L95A Ser, L95B Gly, L95C Ser and L96Gly；Epitope residues 132Gly is interacted with least one be selected from the paratope residue of the following group：H58Tyr and L95A Ser；Epitope residues 133Asn and paratope residue L95A Ser interact；Epitope residues 134Met with least One is selected from the paratope residue interaction with the following group：L93Ser, L94Ser and L95A Ser；And epitope residues 138Glu is interacted with least one be selected from the paratope residue of the following group：L28Gly, L29Ala and L93Ser.
The antibody or its antigen-binding fragment of the present invention can be any and include and at least one above-listed epitope residues phase interaction The antibody or antigen-binding fragment of any of the above-described paratope residue.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include at least one, at least 2 A, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, At least 12, at least 13 or all above-mentioned paratope residues, or times of above-mentioned various types of other paratope residue What is combined.In addition, conservative replacement can be introduced in these paratope residues.For example, antibody or its antigen-binding fragment can Include the conservative replacement 1,2,3,4,5,6,7 or 8 according to table 34.
34 conservative replacement of table
Residue Conservative replacement Residue Conservative replacement
Ala Ser Leu Ile,Val
Arg Lys Lys Arg,Gln
Asn Gln；His Met Leu,Ile
Asp Glu Phe Met,Leu,Tyr
Cys Ser Ser Thr；Gly
Gln Asn Thr Ser,Val
Glu Asp Trp Tyr
Gly Pro Tyr Trp,Phe
His Asn,Gln Val Ile,Leu
Ile Leu,Val Pro -
The antibody of the present invention can have the ability to be combined TFPI of the present invention with another antibody competition as described herein.For example, this The antibody of invention can be resisted with TFPI-23 described in the invention and its variation cross competition combination TFPI, or combination by TFPI-23 The suitable fragment or variation for the TFPI that body combines.This cross-competing antibodies can according to its in standard combination mensuration with this hair The ability of bright exemplary antibodies cross competition is identified.Such as SPR can be used (such as by using BiacoreTMSystem), ELISA is measured or flow cytometry proves cross competition.This cross competition may imply that both antibody bindings are identical, again Folded or similar epitope.
Therefore, antibody of the invention can be identified by the method comprising combination mensuration, and whether this method assessment test antibody Can be with exemplary antibodies (such as TFPI-23 described in the invention or its any variation or fragment) competition binding target of the present invention Binding site on molecule.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include following heavy chain CDR sequences Row：(i) SEQ ID NO are included：38 CDR-H1, include SEQ ID NO：39 CDR-H2 and include SEQ ID NO：40 CDR-H3；And/or (ii) following CDR sequence：Include SEQ ID NO：33 CDR-L1, include SEQ ID NO：34 CDR-L2 and include SEQ ID NO：35 CDR-L3.In certain embodiments, antibody described in the invention or its antigen Binding fragment includes following heavy CDR sequences：(i) with SEQ ID NO：38 at least 85%, at least 90% or at least 95% are identical CDR-H1, with SEQ ID NO：The identical CDR-H2 of 39 at least 85%, at least 90% or at least 95% and with SEQ ID NO： The identical CDR-H3 of 40 at least 85%, at least 90% or at least 95%；And/or (ii) following CDR sequence：With SEQ ID NO：The identical CDR-L1 of 33 at least 85%, at least 90% or at least 95% and SEQ ID NO：34 at least 85%, at least 90% Or at least 95% identical CDR-L2 and with SEQ ID NO：The identical CDR- of 35 at least 85%, at least 90% or at least 95% L3.In certain embodiments, relative to SEQ ID NO：38th, 39,40,33,34 and 35, manufacture in each CDR do not surpass respectively Cross 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, Substitute no more than 2 or no more than 1.In certain embodiments, which is the conservative replacement according to table 34.In some realities Apply in scheme, which is according to the row of table 29A the 4th, the 5th row or the 6th row.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include human framework sequence. For example, heavy chain framework sequence may be from mankind VH3 system genitales, VH1 system genitales, VH5 system genitales or VH4 system genitales.It is preferred that the mankind System genitale heavy chain framework is the framework derived from VH1, VH3 or VH5 system genitale.For example, the VH from following system genitale can be used Framework：IGHV3-23, IGHV3-7 or IGHV1-69 (system genitale title is defined based on IMGT system genitales).It is preferred that human reproduction It is that light chain framework is the framework derived from VK or V λ system genitales.For example, the VL frameworks from following system genitale can be used：IGKV1- 39 or IGKV3-20 (system genitale title is defined based on IMGT system genitales).Additionally or alternatively, frame sequence can be human reproduction The shared frame sequence of system, such as 1 consensus sequences of mankind V λ, VK1 consensus sequences, VK2 consensus sequences, VK3 consensus sequences, VH3 life Grow be consensus sequence, VH1 system genitales consensus sequence, VH5 system genitales consensus sequence or VH4 system genitale consensus sequences framework.
The sequence of human reproduction system framework can be obtained from various disclosed databases, for example, V-base, IMGT, NCBI or Abysis。
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) VH, it includes With selected from the amino acid sequence of the following group at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%th, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, At least 98%, at least 99% or 100% identical amino acid sequence：SEQ ID NO：41st, 63 and 65；And/or (ii) VL, it is wrapped Containing with SEQ ID NO：36 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%th, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence.Any combinations of these VL and VH sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) CH, it includes With SEQ ID NO：20 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) CL, it includes with SEQ ID NO：26 at least 50%, at least 60%th, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence. Any combinations of these CH and CL sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include Fc domains.The Fc Domain can be derived from IgA (such as IgA1Or IgA2), IgG, IgE or IgG (such as IgG1、IgG2、IgG3Or IgG4)。
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) heavy chain, it is wrapped Containing with SEQ ID NO：42、SEQ ID NO：64 or SEQ ID NO：66 at least 50%, at least 60%, at least 70%, at least 75%th, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, At least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) light chain, it is wrapped Containing with SEQ ID NO：37 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%th, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence.Any combinations of these heavy chains and sequence of light chain are also contained in the present invention.
TFPI-24 and variation
Eutectic structure shows that TFPI-24 (and its variation) and TFPI-23 shares many epitope residues.Wherein, Ile105, Arg107 and Leu131 (are numbered, such as SEQ ID NO according to mankind TFPI：Shown in 2) be considered be to antibody-antigen interactions Important.Other shared epitope residues include：Cys106, Gly108, C130, L131 and G132 are (according to SEQ ID NO：2 compile Number).
TFPI-24 (and its variation) special epitope residues are included：Glu100, Glu101, Asp102, Gly104 and Tyr109.TFPI-23 and its variation are not combined with these residues.Referring to table 27.Therefore, the present invention provides specific binding TFPI K2 in epitope separated antibody or its antigen-binding fragment, wherein the epitope (i) include residue Ile105, Arg107 and Leu131；(ii) optionally it is selected from comprising one or more with the residue of the following group：Cys106, Gly108, Cys130, Leu131 and Gly132；And (iii) is optionally further selected from the residue of the following group comprising one or more：Glu100、Glu101、Asp102、 Gly104 and Tyr109 are (according to SEQ ID NO：2 numberings).
In certain embodiments, which does not include one or more and is selected from the residue of the following group：P103、T111、Y113、 F114, N116, Q118, Q121, C122, E123, R124, F125, K126, L140 (are numbered according to SEQ ID NO：2) and its appoint What is combined.Referring to table 27.According to WO201007269 (Novo Nordisk Co., Ltd), reference antibody 4F36 identifications include following epitope： P103, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126 and L140.
In certain embodiments, which does not include one or more and is selected from the residue of the following group：D31、D32、P34、 C35, K36, P103, K126, Y127, G128 are (according to SEQ ID NO：2 numberings) and its any combinations.Referring to table 27.According to table 27, reference antibody 2A8 and 2A8-200 identification include following epitope：D31、D32、P34、C35、K36、P103、K126、Y127 And G128.
Also the paratope residue (being based on BSA) (referring to table 24) from TFPI-24 is characterized and including following： H33Ala、H35Gln、H52Ser、H53Asn、H55Arg、H56Ser、H95Phe、H96Leu、H97His、H99Ser、 H101Asp, L31Met, L32Tyr, L34His, L36Tyr, L50Arg, L91Trp and L96Tyr.In certain embodiments, originally Invent described antibody or its antigen-binding fragment include at least eight, at least nine, at least ten, at least 11, at least 12 A, at least 13, at least 14, at least 15, at least 16, at least 17 or all these paratope residues.In addition, Conservative replacement can be introduced in these paratope residues.For example, the antibody or its antigen-binding fragment can be included according to table 34 1,2,3,4,5,6,7 or 8 conservative replacement.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include following heavy chain CDR sequences Row：(i) SEQ ID NO are included：48 CDR-H1, include SEQ ID NO：49 CDR-H2 and include SEQ ID NO：50 CDR-H3；And/or (ii) following CDR sequence：Include SEQ ID NO：43 CDR-L1, include SEQ ID NO：44 CDR-L2 and include SEQ ID NO：45 CDR-L3.In certain embodiments, antibody described in the invention or its antigen Binding fragment includes following heavy CDR sequences：(i) with SEQ ID NO：48 at least 85%, at least 90% or at least 95% are identical CDR-H1, with SEQ ID NO：The identical CDR-H2 of 49 at least 85%, at least 90% or at least 95% and with SEQ ID NO： The identical CDR-H3 of 50 at least 85%, at least 90% or at least 95%；And/or (ii) following CDR sequence：With SEQ ID NO：The identical CDR-L1 of 43 at least 85%, at least 90% or at least 95% and SEQ ID NO：44 at least 85%, at least 90% Or at least 95% identical CDR-L2 and with SEQ ID NO：The identical CDR- of 45 at least 85%, at least 90% or at least 95% L3.In certain embodiments, relative to SEQ ID NO：48th, 49,50,43,44 and 45, manufacture in each CDR do not surpass respectively Cross 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, Substitute no more than 2 or no more than 1.In certain embodiments, which is the conservative replacement according to table 34.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include human framework sequence. For example, heavy chain framework sequence may be from mankind VH3 system genitales, VH1 system genitales, VH5 system genitales or VH4 reproductions as described above System.It is preferred that human reproduction system light chain framework is the framework as described above for being derived from VK or V λ system genitales.As above institute can also be used The shared mankind's germline framework sequence stated.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include (i) VH, it includes with Selected from the amino acid sequence of the following group at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%th, at least 99% or 100% identical amino acid sequence：SEQ ID NO：67th, 69,51 and 79；And/or (ii) VL, it is wrapped Containing with selected from the amino acid sequence of the following group at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%th, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, At least 98%, at least 99% or 100% identical amino acid sequence：SEQ ID NO：46th, 71,73,75 and 77.These VL and VH Any combinations of sequence are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) CH, it includes With SEQ ID NO：20 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) CL, it includes with SEQ ID NO：26 at least 50%, at least 60%th, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence. Any combinations of these CH and CL sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include Fc domains.The Fc Domain can be derived from IgA (such as IgA1Or IgA2), IgG, IgE or IgG (such as IgG1、IgG2、IgG3Or IgG4)。
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) heavy chain, it is wrapped Containing with SEQ ID NO：52、SEQ ID NO：68、SEQ ID NO：70 or SEQ ID NO：80 at least 50%, at least 60%, extremely Few 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%th, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；With/ Or (ii) light chain, it includes with SEQ ID NO：47、SEQ ID NO：72、SEQ ID NO：74、SEQ ID NO：76 or SEQ ID NO：78 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%th, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or 100% identical amino acid sequence.Any combinations of these heavy chains and sequence of light chain are also contained in the disclosure.
4D8 and variation
Eutectic structure also discloses the epitope and paratope information of antibody 4D8 and variation.The epitope of 4D8 and its variation is residual Base includes：Glu101, Pro103, Tyr109, Thr111, Ser119, Gln121, Glu123, Arg124, Lys126 and Leu140 (according to SEQ ID NO：2 numberings).
In certain embodiments, which does not include one or more and is selected from the residue of the following group：E100、D102、R107、 Y113, F114, N116, Q118, C122 are (according to SEQ ID NO：2 numberings) and its any combinations.Referring to table 27.According to WO201007269 (Novo Nordisk Co., Ltd), reference antibody 4F36 identification comprising E100, D102, R107, Y113, F114, N116, The epitope of Q118 and C122.
In certain embodiments, which does not include one or more and is selected from the residue of the following group：D31、D32、P34、 C35, K36, E100, I105, R107, G108, Y127, G128 are (according to SEQ ID NO：2 numberings) and its any combinations.Referring to table 27.According to table 27, reference antibody 2A8 and 2A8-200 identification comprising D31, D32, P34, C35, K36, P103, K126, Y127 and The epitope of G128.
Also the paratope residue (being based on BSA) (referring to table 24) from 4D8 is characterized and including following：H50Asp、 H57Thr、H58Leu、H59Tyr、H61Gln、H98Asp、H99Tyr、H100Asp、L30His、L50Trp、L92Tyr、 L93Thr, L94Thr and L96Tyr.In certain embodiments, antibody described in the invention or its antigen-binding fragment include At least eight, at least nine, at least ten, at least at least 11, at least 12,13 or all these paratope residues. In addition, conservative replacement can be introduced in these paratope residues.For example, the antibody or its antigen-binding fragment can include basis 1,2,3,4,5,6,7 or 8 conservative replacement of table 34.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include following heavy chain CDR sequences Row：(i) SEQ ID NO are included：87 CDR-H1, include SEQ ID NO：88 CDR-H2 and include SEQ ID NO：89 CDR-H3；And/or (ii) following CDR sequence：Include SEQ ID NO：81 CDR-L1, include SEQ ID NO：82 CDR-L2 and include SEQ ID NO：83 CDR-L3.In certain embodiments, antibody described in the invention or its antigen Binding fragment includes following heavy CDR sequences：(I) and SEQ ID NO：87 at least 85%, at least 90% or at least 95% are identical CDR-H1, with SEQ ID NO：The identical CDR-H2 of 88 at least 85%, at least 90% or at least 95% and with SEQ ID NO： The identical CDR-H3 of 89 at least 85%, at least 90% or at least 95%；And/or (ii) following CDR sequence：With SEQ ID NO：The identical CDR-L1 of 81 at least 85%, at least 90% or at least 95% and SEQ ID NO：82 at least 85%, at least 90% Or at least 95% identical CDR-L2 and with SEQ ID NO：The identical CDR- of 83 at least 85%, at least 90% or at least 95% L3.In certain embodiments, relative to SEQ ID NO：87th, 88,89,81,82 and 83, manufacture in each CDR do not surpass respectively Cross 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, Substitute no more than 2 or no more than 1.In certain embodiments, which is the conservative replacement according to table 34.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include human framework sequence. For example, heavy chain framework sequence may be from mankind VH3 system genitales, VH1 system genitales, VH5 system genitales or VH4 reproductions as described above System.It is preferred that human reproduction system light chain framework is the framework derived from VK or V λ system genitales as described above.As above institute can also be used The shared mankind's germline framework sequence stated.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) VH, it includes With selected from the amino acid sequence of the following group at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%th, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, At least 98%, at least 99% or 100% identical amino acid sequence：SEQ ID NO：90th, 95,97,99,101,103,105 and 107；And/or (ii) VL, it includes with selected from the amino acid sequence of the following group at least 50%, at least 60%, at least 70%, at least 75%th, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, At least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence：SEQ ID NO：84th, 109 and 111.Any combinations of these VL and VH sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) CH, it includes With SEQ ID NO：20 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) CL, it includes with SEQ ID NO：91 or SEQ ID NO：85 to Few 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%th, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% phase Same amino acid sequence.Any combinations of these CH and CL sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include Fc domains.The Fc Domain can be derived from IgA (such as IgA1Or IgA2), IgG, IgE or IgG (such as IgG1、IgG2、IgG3Or IgG4)。
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) heavy chain, it is wrapped Containing with SEQ ID NO：92、SEQ ID NO：94、SEQ ID NO：96、SEQ ID NO：98、SEQ ID NO：100、SEQ ID NO：102、SEQ ID NO：104、SEQ ID NO：106、SEQ ID NO：108 at least 50%, at least 60%, at least 70%, extremely Few 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%th, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) is light Chain, it includes with SEQ ID NO：86、SEQ ID NO：93、SEQ ID NO：110 or SEQ ID NO：112 at least 50%, extremely Few 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid Sequence.Any combinations of these heavy chains and sequence of light chain are also contained in the present invention.
TFPI-3 and variation
The present invention also provides TFPI-3 and its variation.Therefore, included based on the antibody of TFPI-3 or its antigen-binding fragment Following heavy CDR sequences：(i) SEQ ID NO are included：16 CDR-H1, include SEQ ID NO：17 CDR-H2 and include SEQ ID NO：18 CDR-H3；And/or (ii) following CDR sequence：Include SEQ ID NO：10 CDR-L1, include SEQ ID NO：11 CDR-L2 and include SEQ ID NO：12 CDR-L3.In certain embodiments, antibody described in the invention or Its antigen-binding fragment includes following heavy CDR sequences：(i) with SEQ ID NO：16 at least 85%, at least 90% or at least 95% identical CDR-H1 and SEQ ID NO：17 at least 85%, at least 90% or at least 95% identical CDR-H2 and and SEQ ID NO：The identical CDR-H3 of 18 at least 85%, at least 90% or at least 95%；And/or (ii) following CDR sequence：With SEQ ID NO：The identical CDR-L1 of 10 at least 85%, at least 90% or at least 95% and SEQ ID NO：11 at least 85%, extremely Few 90% or at least 95% identical CDR-L2 and with SEQ ID NO：12 at least 85%, at least 90% or at least 95% are identical CDR-L3.In certain embodiments, relative to SEQ ID NO：16th, 17,18,10,11 and 12, manufactured respectively in each CDR No more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, be no more than 3, no more than 2 or no more than 1 substitution.In certain embodiments, which is the conservative replacement according to table 34.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include human framework sequence. For example, heavy chain framework sequence may be from mankind VH3 system genitales, VH1 system genitales, VH5 system genitales or VH4 reproductions as described above System.It is preferred that human reproduction system light chain framework is derived from the framework for being as above VK the or V λ system genitales stated.As above institute can also be used The shared mankind's germline framework sequence stated.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) VH, it includes With SEQ ID NO：19 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and/or (ii) VL, it includes with SEQ ID NO：13 at least 50%, at least 60%th, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence. Any combinations of these VL and VH sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) CH, it includes With SEQ ID NO：20 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) CL, it includes with SEQ ID NO：91 or SEQ ID NO：14 to Few 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%th, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% phase Same amino acid sequence.Any combinations of these CH and CL sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include Fc domains.The Fc Domain can be derived from IgA (such as IgA1Or IgA2), IgG, IgE or IgG (such as IgG1、IgG2、IgG3Or IgG4)。
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) heavy chain, it is wrapped Containing with SEQ ID NO：21 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%th, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence；And/or (ii) light chain, it includes with SEQ ID NO：15 at least 50%, extremely Few 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid Sequence.Any combinations of these heavy chains and sequence of light chain are also contained in the present invention.
TFPI-21 and variation
The present invention also provides TFPI-21 and its variation.Therefore, based on the antibody of TFPI-21 or its antigen-binding fragment bag Containing following heavy CDR sequences：(i) SEQ ID NO are included：28 CDR-H1, include SEQ ID NO：29 CDR-H2 and comprising SEQ ID NO：30 CDR-H3；And/or (ii) following CDR sequence：Include SEQ ID NO：22 CDR-L1, include SEQ ID NO：23 CDR-L2 and include SEQ ID NO：24 CDR-L3.In certain embodiments, it is described in the invention Antibody or its antigen-binding fragment include following heavy CDR sequences：(i) with SEQ ID NO：28 at least 85%, at least 90% Or at least 95% identical CDR-H1, with SEQ ID NO：The identical CDR-H2 of 29 at least 85%, at least 90% or at least 95% With with SEQ ID NO：The identical CDR-H3 of 30 at least 85%, at least 90% or at least 95%；And/or (ii) following light chain CDR Sequence：With SEQ ID NO：The identical CDR-L1 of 22 at least 85%, at least 90% or at least 95% and SEQ ID NO：23 at least 85%th, at least 90% or at least 95% identical CDR-L2 and with SEQ ID NO：24 at least 85%, at least 90% or at least 95% identical CDR-L3.In certain embodiments, relative to SEQ ID NO：28th, 29,30,22,23 and 24, respectively each In CDR manufacture no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4 A, no more than 3, no more than 2 or no more than 1 substitution.In certain embodiments, which is according to the conservative of table 34 Substitution.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include human framework sequence. For example, heavy chain framework sequence may be from mankind VH3 system genitales, VH1 system genitales, VH5 system genitales or VH4 reproductions as described above System.It is preferred that human reproduction system light chain framework is the framework as described above for being derived from VK or V λ system genitales.It can also be used above-mentioned Shared mankind's germline framework sequence.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) VH, it includes With SEQ ID NO：31 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and/or (ii) VL, it includes with SEQ ID NO：25 at least 50%, at least 60%th, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence. Any combinations of these VL and VH sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) CH, it includes With SEQ ID NO：20 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) CL, it includes with SEQ ID NO：91 or SEQ ID NO：26 to Few 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%th, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% phase Same amino acid sequence.Any combinations of these CH and CL sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include Fc domains.The Fc Domain can be derived from IgA (such as IgA1Or IgA2), IgG, IgE or IgG (such as IgG1、IgG2、IgG3Or IgG4)。
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) heavy chain, it is wrapped Containing with SEQ ID NO：32 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%th, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence；And/or (ii) light chain, it includes with SEQ ID NO：27 at least 50%, extremely Few 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid Sequence.Any combinations of these heavy chains and sequence of light chain are also contained in the present invention.
TFPI-26 and variation
The present invention also provides TFPI-26 and its variation.Therefore, based on the antibody of TFPI-26 or its antigen-binding fragment bag Containing following heavy CDR sequences：(i) SEQ ID NO are included：58 CDR-H1, include SEQ ID NO：59 CDR-H2 and comprising SEQ ID NO：60 CDR-H3；And/or (ii) following CDR sequence：Include SEQ ID NO：53 CDR-L1, include SEQ ID NO：54 CDR-L2 and include SEQ ID NO：55 CDR-L3.In certain embodiments, it is described in the invention Antibody or its antigen-binding fragment include following heavy CDR sequences：(i) with SEQ ID NO：58 at least 85%, at least 90% Or at least 95% identical CDR-H1, with SEQ ID NO：The identical CDR-H2 of 59 at least 85%, at least 90% or at least 95% With with SEQ ID NO：The identical CDR-H3 of 60 at least 85%, at least 90% or at least 95%；And/or (ii) following light chain CDR Sequence：With SEQ ID NO：The identical CDR-L1 of 53 at least 85%, at least 90% or at least 95% and SEQ ID NO：54 at least 85%th, at least 90% or at least 95% identical CDR-L2 and with SEQ ID NO：55 at least 85%, at least 90% or at least 95% identical CDR-L3.In certain embodiments, relative to SEQ ID NO：58th, 59,60,53,54 and 55, respectively each In CDR manufacture no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4 A, no more than 3, no more than 2 or no more than 1 substitution.In certain embodiments, which is according to the conservative of table 34 Substitution.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include human framework sequence. For example, heavy chain framework sequence may be from mankind VH3 system genitales, VH1 system genitales, VH5 system genitales or VH4 reproductions as described above System.It is preferred that human reproduction system light chain framework is the framework as described above for being derived from VK or V λ system genitales.As above institute can also be used The shared mankind's germline framework sequence stated.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) VH, it includes With SEQ ID NO：61 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and/or (ii) VL, it includes with SEQ ID NO：56 at least 50%, at least 60%th, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence. Any combinations of these VL and VH sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) CH, it includes With SEQ ID NO：20 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) CL, it includes with SEQ ID NO：26 at least 50%, at least 60%th, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence. Any combinations of these CH and CL sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include Fc domains.The Fc Domain can be derived from IgA (such as IgA1Or IgA2), IgG, IgE or IgG (such as IgG1、IgG2、IgG3Or IgG4)。
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) heavy chain, it is wrapped Containing with SEQ ID NO：62 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%th, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence；And/or (ii) light chain, it includes with SEQ ID NO：57 at least 50%, extremely Few 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid Sequence.Any combinations of these heavy chains and sequence of light chain are also contained in the present invention.
6B7.c5 and variation
The present invention also provides 6B7.c5 and its variation.Therefore, included based on the antibody of 6B7.c5 or its antigen-binding fragment Following heavy CDR sequences：(i) SEQ ID NO are included：118 CDR-H1, include SEQ ID NO：119 CDR-H2 and comprising SEQ ID NO：120 CDR-H3；And/or (ii) following CDR sequence：Include SEQ ID NO：113 CDR-L1, include SEQ ID NO：114 CDR-L2 and include SEQ ID NO：115 CDR-L3.In certain embodiments, the present invention is retouched The antibody stated or its antigen-binding fragment include following heavy CDR sequences：(i) with SEQ ID NO：118 at least 85%, at least 90% or at least 95% identical CDR-H1 and SEQ ID NO：119 at least 85%, at least 90% or at least 95% are identical CDR-H2 and with SEQ ID NO：The identical CDR-H3 of 120 at least 85%, at least 90% or at least 95%；And/or (ii) is following CDR sequence：With SEQ ID NO：The identical CDR-L1 of 113 at least 85%, at least 90% or at least 95% and SEQ ID NO：The identical CDR-L2 of 114 at least 85%, at least 90% or at least 95% and with SEQ ID NO：115 at least 85%, at least 90% or at least 95% identical CDR-L3.In certain embodiments, relative to SEQ ID NO：118、119、120、113、 114 and 115, respectively in each CDR manufacture no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, No more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 substitution.In certain embodiments, this takes Generation is the conservative replacement according to table 34.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include human framework sequence. For example, heavy chain framework sequence may be from mankind VH3 system genitales, VH1 system genitales, VH5 system genitales or VH4 reproductions as described above System.It is preferred that human reproduction system light chain framework is the framework as described above for being derived from VK or V λ system genitales.As above institute can also be used The shared mankind's germline framework sequence stated.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) VH, it includes With SEQ ID NO：121 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%th, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence, and/or (ii) VL, it includes with SEQ ID NO：116 at least 50%, extremely Few 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid Sequence.Any combinations of these VL and VH sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) CH, it includes With SEQ ID NO：91 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) CL, it includes with SEQ ID NO：91 or SEQ ID NO：85 to Few 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%th, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% phase Same amino acid sequence.Any combinations of these CH and CL sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include Fc domains.The Fc Domain can be derived from IgA (such as IgA1Or IgA2), IgG, IgE or IgG (such as IgG1、IgG2、IgG3Or IgG4)。
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) heavy chain, it is wrapped Containing with SEQ ID NO：122 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%th, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence；And/or (ii) light chain, it includes with SEQ ID NO：117 at least 50%, At least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid Sequence.Any combinations of these heavy chains and sequence of light chain are also contained in the present invention.
7A4.D9 and variation
The present invention also provides 7A4.D9 and its variation.Therefore, included based on the antibody of 7A4.D9 or its antigen-binding fragment Following heavy CDR sequences：(i) SEQ ID NO are included：128 CDR-H1, include SEQ ID NO：129 CDR-H2 and comprising SEQ ID NO：130 CDR-H3；And/or (ii) following CDR sequence：Include SEQ ID NO：123 CDR-L1, include SEQ ID NO：124 CDR-L2 and include SEQ ID NO：125 CDR-L3.In certain embodiments, the present invention is retouched The antibody stated or its antigen-binding fragment include following heavy CDR sequences：(i) with SEQ ID NO：128 at least 85%, at least 90% or at least 95% identical CDR-H1 and SEQ ID NO：129 at least 85%, at least 90% or at least 95% are identical CDR-H2 and with SEQ ID NO：The identical CDR-H3 of 130 at least 85%, at least 90% or at least 95%；And/or (ii) is following CDR sequence：With SEQ ID NO：The identical CDR-L1 of 123 at least 85%, at least 90% or at least 95% and SEQ ID NO：The identical CDR-L2 of 124 at least 85%, at least 90% or at least 95% and with SEQ ID NO：125 at least 85%, at least 90% or at least 95% identical CDR-L3.In certain embodiments, relative to SEQ ID NO：128、129、130、123、 124 and 125, respectively in each CDR manufacture no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, No more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 substitution.In certain embodiments, this takes Generation is the conservative replacement according to table 34.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include human framework sequence. For example, heavy chain framework sequence may be from mankind VH3 system genitales, VH1 system genitales, VH5 system genitales or VH4 reproductions as described above System.It is preferred that human reproduction system light chain framework is the framework as described above for being derived from VK or V λ system genitales.As above institute can also be used The shared mankind's germline framework sequence stated.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) VH, it includes With SEQ ID NO：131 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%th, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence, and/or (ii) VL, it includes with SEQ ID NO：126 at least 50%, extremely Few 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid Sequence.Any combinations of these VL and VH sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) CH, it includes With SEQ ID NO：91 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence；And/or (ii) CL, it includes with SEQ ID NO：91 or SEQ ID NO：85 to Few 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%th, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% phase Same amino acid sequence.Any combinations of these CH and CL sequences are also contained in the present invention.
In certain embodiments, antibody described in the invention or its antigen-binding fragment include Fc domains.The Fc Domain can be derived from IgA (such as IgA1Or IgA2), IgG, IgE or IgG (such as IgG1、IgG2、IgG3Or IgG4)。
In certain embodiments, antibody described in the invention or its antigen-binding fragment include：(i) heavy chain, it is wrapped Containing with SEQ ID NO：132 at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%th, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence；And/or (ii) light chain, it includes with SEQ ID NO：127 at least 50%, At least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%th, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid Sequence.Any combinations of these heavy chains and sequence of light chain are also contained in the present invention.
Also disclose that specific binding TFPI K2 domains and with any antibody described in the invention or its antigen knot Close the antibody or its antigen knot of fragment (such as any antibody (or its antigen-binding fragment) listed in table 3) competition binding TFPI Close fragment.For example, if the combination of antibody or its antigen-binding portion thereof and TFPI hinder follow-up TFPI-23 or TFPI-106 and TFPI With reference to the then antibody or its antigen-binding portion thereof and TFPI-23 or TFPI-106 competition bindings TFPI.
Also disclose that specific binding TFPI K2 domains and with any antibody described in the invention or its antigen knot Close fragment (such as in table 3 listed any antibody (or its antigen-binding fragment)) be bound to identical TFPI epitopes antibody or Its antigen-binding fragment.
It is provided in by the exemplary antibodies competition assay (and overlapping epitope analysis) of SPR in embodiment 6.
Antibody and antigen-binding fragment disclosed in this invention include monoclonal antibody, polyclonal antibody, antibody fragment (example Such as Fab, Fab', F (ab')2, Fv, Fc, etc.), chimeric antibody, bispecific antibody, Heteroconjugate antibodies (heteroconjugate antibody), single-stranded (ScFv), its mutant, the fusion protein comprising antibody moiety, domain Any other of antibody (dAb), humanized antibody and the immunoglobulin molecules comprising required specific antigen recognition site are repaiied The configuration (antibody for including the glycosylation variants of antibody, the amino acid sequence variation of antibody and covalent modification) of decorations.Antibody and anti- Former binding fragment can be mouse, rat, the mankind or any other source (including chimeric or humanized antibody).In some embodiment party In case, which is monoclonal antibody.In some embodiments, the antibody be fitted together to, humanization or human antibodies.Some In embodiment, which is human antibodies.In certain embodiments, which is humanized antibody.
In certain embodiments, compatibility (Kd) value of antibody and its antigen-binding fragment disclosed in this invention does not surpass Cross about 1 × 10-3M, such as no more than about 5 × 10-4M, 4 × 10 are no more than about-4M, 3 × 10 are no more than about-4M, be no more than about 2 × 10-4M, it is no more than about 1 × 10-4M, 9 × 10 are no more than about-5M, 8 × 10 are no more than about-5M, 7 × 10 are no more than about-5M, it is no more than About 6 × 10-5M, 5 × 10 are no more than about-5M, 4 × 10 are no more than about-5M, 3 × 10 are no more than about-5M, 2 × 10 are no more than about-5M、 No more than about 1 × 10-5M, 9 × 10 are no more than about-6M, 8 × 10 are no more than about-6M, 7 × 10 are no more than about-6M, be no more than about 6 × 10-6M, 5 × 10 are no more than about-6M, 4 × 10 are no more than about-6M, 3 × 10 are no more than about-6M, 2 × 10 are no more than about-6M, it is no more than About 1 × 10-6M, 9 × 10 are no more than about-7M, 8 × 10 are no more than about-7M, 7 × 10 are no more than about-7M, 6 × 10 are no more than about-7M、 No more than about 5 × 10-7M, 4 × 10 are no more than about-7M, 3 × 10 are no more than about-7M, 2 × 10 are no more than about-7M, be no more than about 1 × 10-7M, 9 × 10 are no more than about-8M, 8 × 10 are no more than about-8M, 7 × 10 are no more than about-8M, 6 × 10 are no more than about-8M, it is no more than About 5 × 10-8M, 4 × 10 are no more than about-8M, 3 × 10 are no more than about-8M, 2 × 10 are no more than about-8M, it is no more than about 1 × 10-8M、 No more than about 9 × 10-9M, 8 × 10 are no more than about-9M, 7 × 10 are no more than about-9M, 6 × 10 are no more than about-9M, be no more than about 5 × 10-9M, 4 × 10 are no more than about-9M, 3 × 10 are no more than about-9M, 2 × 10 are no more than about-9M, it is no more than about 1 × 10-9M, about 1 × 10-3To about 1 × 10-13M、1×10-4To about 1 × 10-13M、1×10-5To about 1 × 10-13M, about 1 × 10-6To about 1 × 10-13M、 About 1 × 10-7To about 1 × 10-13M, about 1 × 10-8To about 1 × 10-13M, about 1 × 10-9To about 1 × 10-13M、1×10-3To about 1 × 10-12M、1×10-4To about 1 × 10-12M, about 1 × 10-5To about 1 × 100-12M, about 1 × 10-6To about 1 × 10-12M, about 1 × 10-7 To about 1 × 10-12M, about 1 × 10-8To about 1 × 10-12M, about 1 × 10-9To about 1 × 10-12M、1×10-3To about 1 × 10-11M、1 ×10-4To about 1 × 10-11M, about 1 × 10-5To about 1 × 10-11M, about 1 × 10-6To about 1 × 10-11M, about 1 × 10-7To about 1 × 10-11M, about 1 × 10-8To about 1 × 10-11M, about 1 × 10-9To about 1 × 10-11M、1×10-3To about 1 × 10-10M、1×10-4Extremely About 1 × 10-10M, about 1 × 10-5To about 1 × 10-10M, about 1 × 10-6To about 1 × 10-10M, about 1 × 10-7To about 1 × 10-10M, about 1×10-8To about 1 × 10-10M or about 1 × 10-9To about 1 × 10-10M。
In certain embodiments, dissociation constant is measured using surface plasma body resonant vibration (SPR) method (Biacore).Table Surface plasma resonance refers to a kind of optical phenomena, it is for example by using BIACORETMSystem detectio biology sensor matrix The change of interior protein concentration interacts so as to analyze biologic specificity in real time.In certain embodiments, which measures Carried out using Biacore T100 or T200 instrument.
For example, the standard assay conditions for surface plasma body resonant vibration can be based on about 100 IgG reactons (RU) Ligand is fixed on SPR chips.The target protein of purifying is diluted to ultimate density scope and with required flow velocity (example with buffer Such as 10-100 μ l/min) injection, to calculate Ka.Dissociation is carried out to establish dissociation rate (Kd), then carry out 5 seconds 3M MgCl2 (or 20mM NaOH) pulse is so that chip surface regenerates.Then, analyzed and sensed using kinetic evaluation software bag (sensorgram)。
In an exemplary embodiment, SPR measure is according to subtitle " surface plasma body resonant vibration in embodiment 1 (SPR) " listed condition under.
In certain embodiments, dissociation constant excludes measure (KinExA using the dynamics based on solutionTM) measurement. In particular, KinExA measurements use KinExATM3200 instruments (Sapidyne) carry out.The dynamics excludes measure (KinExATM) be can measure antigen/antibody interaction equilibrium dissociation constant and association and dissociation speed constant it is general Immunoassays platform (substantially streaming fluorescence spectrophotometer fluorescence photometer (flow spectrofluorometer)).Due to KinExATM Carried out after having been balanced, its be for measure high-affinity interaction Kd Advantageous techniques, the wherein interaction Dissociation rate may be very slow.The KinExATMMethod can be substantially according to Drake et al (2004) Analytical Description in Biochemistry 328,35-43 carries out.
In general, TFPI antibody is needed with high-affinity combination TFPI effectively to block the activity of TFPI.But by Also expressed in TFPI on cell surface, when the binding affinity of the antibody is excessive, the antibody can soon by internalization and by Host cell is degraded.This can potentially result in short half-life period and duplicate injection.For example, compared with TFPI-24, antibody TFPI- 23 show relatively low binding affinity (Kd), and are even more ideal in some cases, because it is with relatively low internalization speed Rate and longer half-life period.Therefore, if desired longer half-life period, then binding affinity (Kd) is 5 × 10-7M to about 5 × 10- 11M, especially about 1 × 10-8To about 1 × 10-10M (0.1nM to 10nM) is generally desired.This scope is considered effective at (i) Suppress to be put down between the active required binding affinity of TFPI, and (ii) longer half-life period and reduction antibody internalization Weighing apparatus.
Especially, it is believed that in order to maintain weekly with 3mg/kg subcutaneous administrations, Kd values are about 1 × 10-8M to about 1 × 10-10M (0.1nM to 10nM) is desired.
Whether antibody or its antigen-binding fragment reduce the activity of TFPI or reduce TFPI is tied with physiologic substrate (such as FXa) Closing can resisted by measuring the reduction of TFPI and the binding affinity of the physiologic substrate to measure, such as by comparing (i) In the presence of TFPI antibody (or its antigen-binding fragment), the binding affinity of TFPI and its substrate and (ii) in anti-TFPI antibody not In the presence of, the binding affinity of TFPI and same substrate carries out.In the presence of anti-TFPI antibody (or its antigen-binding fragment), The combination of TFPI and physiologic substrate (such as FXa) can reduce at least about 10%, at least about 20%, at least about 30%, at least about 40%th, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%th, at least about 97%, at least about 98% or at least about 99%.When the antibody (or fragment) is not present, TFPI and its physiology The expection combination of substrate can be set to 100%.
Also model and/or external use such as plasma systems anti-TFPI antibody or its antigen-binding fragment can be assessed in vivo TFPI inhibitory activity (in the present invention also referred to as the activity of TFPI " reduce ").For example, antibody inhibitory activity (or reduce TFPI Active level) following assessment can be passed through：(i) reduce measured by the dilution prothrombin time based on blood plasma Clotting time；(ii) whole blood coagulation time as measured by Thrombectomy is reduced；(iii) fibrin ferment generation is increased；(iv) Increase FXa activity in the presence of TFPI；(v) in the presence of TFPI, the platelet accumulation of enhancing；(vi) in the presence of TFPI, increase The fibrin that adds it is raw or；Or (vii) its any combinations.The inhibitory activity of antibody or antigen-binding fragment can be dose dependent (such as causing measured clotting time dose dependent in the dilution prothrombin time based on blood plasma to reduce).
The exemplary mensuration of several TFPI inhibitory activity for assessing antibody is described later in detail in embodiment.Example Such as, diluted plasma prothrombin time (PT) measure is the PT measure of modification, it utilizes diluted factor I (thromboplastin) or tissue factor is to extend the clotting time of the measure and dynamic range.The anti-TFPI of suppression/neutralization resists Body should can reduce dilution prothrombin time.
Another exemplary model system for measuring TFPI inhibitory activity is that exogenous tenase is measured, its test antibody Or its antigen-binding fragment replys the ability activated by exogenous compound-mediated FX in the presence of TFPI.It is another to be used to characterize The model system of TFPI inhibitory activity is that FXa suppresses measure, wherein measuring FXa activity in the presence of TFPI (referring to Sprecher et al.,Proc.Nat.Acad.Sci.USA 91:3353-3357(1994))。
Also antibody or the inhibitory activity of its antigen-binding fragment can be assessed in the measure based on blood plasma.In anti-TFPI antibody Or in the presence of its antigen-binding fragment, can (such as remaining coagulation factor activity be low substantially lacking FVIII or FIX activity Fibrin ferment is triggered in blood plasma 1%) to be formed.Fibrin ferment forms usable fluorogenic substrate or chromogenic substrate detection.Factor Conversion is using such as ThrombographTM(Thermo Scientific, Waltham, Mass.) is measured, and the data obtained can Pass through the Thrombinoscope obtained from Thrombinoscope BVTMThe automatic fibrin ferment generation of software translating generation calibration Scheme (Calibrated Automatic Thrombogram).
For example, antibody or antigen-binding fragment can improve (such as in FVIII exhausts blood plasma) TFPI when FVIII is not present The fibrin ferment generation of adjusting reaches the generation of TFPI dependences fibrin ferment is horizontal in normal plasma at least 1%.It is in general, normal Contain the Factor IX of about 0.5U/mL to about 2U/ml in (no puzzlement) blood plasma.Therefore, in some cases, it is of the invention anti- Body or antigen-binding fragment, which form the fibrin ferment strengthened when FVIII is not present, reaches that there are during 0.5U/ml to 2U/mlFVIII Observed at least about 1%.In a further embodiment, which strengthens FVIII not In the presence of fibrin ferment formed and reach in normal plasma the fibrin ferment of (that is, in the Factor IX presence of physiological level) and form level At least about 2%, at least about 3%, at least about 5%, at least about 7% or at least about 10%.
The antibody or antigen-binding fragment can also be applied to blood coagulation azymia or haemophiliachemophiliac animal model to characterize TFPI Internal inhibitory activity.These In vivo models are known in the art, and including inducing blood friendly for example using the anti-FVIII antibody The mouse (Tranholm et al., Blood, 102,3615-3620 (2003)) of sick A；Clotting factor knockout model, such as but It is not limited to FVIII knock-out mices (Bi et al., Nat.Genet., 10 (1), 119-121 (1995)) and FIX knock-out mices (Wang et al.,Proc.Nat.Acad.Sci.USA 94(21):11563-11566(1997)；The hemophilia A of rabbit induction (Shen et al.,Blood,42(4):509-521(1973))；And Chapel Hill HA dogs (Lozier et al., Proc.Nat.Acad.Sci.USA 99:12991-12996(2002))。
In certain embodiments, antibody (or antigen-binding fragment) disclosed by the invention TFPI there are when strengthen FXa activity, its half maximum effective concentration (EC50) it is no more than 1 × 10-4M, no more than 1 × 10-5M, no more than 1 × 10-6M, do not surpass Cross 1 × 10-7M, no more than 1 × 10-8M, no more than 1 × 10-9M, no more than 1 × 10-10M, no more than 1 × 10-11M or no more than 1 ×10-12M.It is preferred that the EC50It is about 5 × 10-7M to 1 × 10-11M, e.g., from about 1 × 10-7M to 5 × 10-10M, about 1 × 10-7M to 1 ×10-10M、1×10-7M to 5 × 10-9M、5×10-7M to 5 × 10-10M, about 5 × 10-7M to 1 × 10-10M or about 5 × 10-7M is extremely 5×10-9M。
In certain embodiments, antibody of the invention (or antigen-binding fragment) neutralizes what TFPI mediated FVIIa/TF The inhibitory action of FX activation, its half maximum effective concentration (EC50) it is no more than 1 × 10-4M, no more than 1 × 10-5M, no more than 1 × 10-6M, no more than 1 × 10-7M, no more than 1 × 10-8M, no more than 1 × 10-9M, no more than 1 × 10-10M, no more than 1 × 10-11M Or no more than 1 × 10-12M.It is preferred that the EC50It is about 5 × 10-7M to 1 × 10-11M, e.g., from about 1 × 10-7M to 5 × 10-10M, about 1 ×10-7M to 1 × 10-10M、1×10-7M to 5 × 10-9M、5×10-7M to 5 × 10-10M, about 5 × 10-7M to 1 × 10-10M or about 5×10-7M to 5 × 10-9M。
In certain embodiments, as measured in the dilution prothrombin time based on blood plasma, the present invention Disclosed antibody (or antigen-binding fragment) reduces the clotting time, its half maximum effective concentration (EC50) it is no more than 1 × 10-4M, not More than 1 × 10-5M, no more than 1 × 10-6M, no more than 1 × 10-7M, no more than 1 × 10-8M, no more than 1 × 10-9M, it is no more than 1×10-10M, no more than 1 × 10-11M or no more than 1 × 10-12M.It is preferred that the EC50It is about 5 × 10-7M to 1 × 10-11M, such as About 1 × 10-7M to 5 × 10-10M, about 1 × 10-7M to 1 × 10-10M、1×10-7M to 5 × 10-9M、5×10-7M to 5 × 10-10M、 About 5 × 10-7M to 1 × 10-10M or about 5 × 10-7M to 5 × 10-9M。
In certain embodiments, antibody (or antigen-binding fragment) increase fibrin ferment generating rate disclosed by the invention refers to Number, its half maximum effective concentration (EC50) it is no more than 1 × 10-4M, no more than 1 × 10-5M, no more than 1 × 10-6M, no more than 1 × 10-7M, no more than 1 × 10-8M, no more than 1 × 10-9M, no more than 1 × 10-10M, no more than 1 × 10-11M or no more than 1 × 10-12M.It is preferred that the EC50It is about 5 × 10-7M to 1 × 10-11M, e.g., from about 1 × 10-7M to 5 × 10-10M, about 1 × 10-7M to 1 × 10-10M、1×10-7M to 5 × 10-9M、5×10-7M to 5 × 10-10M, about 5 × 10-7M to 1 × 10-10M or about 5 × 10-7M to 5 × 10-9M。
In certain embodiments, antibody and antibody fragment disclosed by the invention also can further pass through other biological activity Measure evaluation, for example, to assess its potency, pharmaceutical active and the potential effect as therapeutic agent.These measure be this area Know and depending on the desired use of the target antigen and the antibody.Example includes such as growth of tumour cell and suppresses measure；Antibody according to Rely the cytotoxicity (CDC) of property cytotoxicity (ADCC) and complement-mediated measure；Agonist activity or antagonistic activity measure.
C. polynucleotides, carrier and host cell
The present invention also provides the polynucleotides of any TFPI binding antibodies disclosed in code book, including described in the invention Antibody fragment and the antibody of modification, for example, as having the antibody of the Fc effector functions weakened.On the other hand, the present invention carries Method for preparing any polynucleotides described in the invention.Polynucleotides can be prepared by methods known in the art and table Reach.Therefore, the present invention provides polynucleotides or composition and (including includes any the TFPI antibody and its antigen knot of the coding present invention Close the pharmaceutical composition of the polynucleotides of fragment).
In one embodiment, VH and VL domains or its antigen-binding fragment or total length HC or LC are by different more Nucleotide coding.Alternatively, both VH and VL or its antigen-binding fragment or HC and LC are by single polynucleotide encoding.
Any the TFPI antibody and its antigen-binding fragment that the present invention provides the coding present invention (include, but are not limited to TFPI- 23rd, TFPI-24, TFPI-106, TFPI-107 and 4D8) polynucleotides or composition comprising polynucleotides, the wherein multinuclear The sequence of thuja acid includes SEQ ID NO：175 (coding TFPI-106VH areas), SEQ ID NO：176 (coding TFPI-106VL Area), SEQ ID NO：177 (coding TFPI-106 heavy chains) and SEQ ID NO：The sequence of 178 (coding TFPI-106 light chains).
On the other hand, the present invention provides VH areas or its antigen-binding portion thereof of the antibody of coding specific binding TFPI Separated nucleic acid, it includes be present in the nucleic acid sequence of the Insert Fragment in the plasmid of ATCC preserving number PTA-122329 preservations Row.
On the other hand, the present invention provides VL areas or its antigen-binding portion thereof of the antibody of coding specific binding TFPI Separated nucleic acid, it includes be present in the nucleic acid sequence of the Insert Fragment in the plasmid of ATCC preserving number PTA-122328 preservations Row.
On the other hand, the present invention provides the polynucleotides and its variation of coding TFPI antibody or part thereof, wherein these Variant polynucleotides include with any specific nucleic acid sequence disclosed by the invention have at least 70%, at least 75%, at least 80%, At least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%th, the nucleotide sequence of at least 96%, at least 97%, at least 98% or at least 99% sequence thereto.The core of any length Sequence thereto degree on nucleotide sequence can utilize method known to those of ordinary skill in the art to calculate.It is unrestricted one Property embodiment in, percentage sequence identity between two or multiple related nucleotide sequences is using from National The nucleotide BLAST servers (http that Library of Medicine are obtained://blast.ncbi.nlm.nih.gov/) survey It is fixed.The software different settings are provided so that those of ordinary skill in the art can according to such as factor of length, complexity and its He compares because of usually optimization.
The present invention provides the nucleic acid molecules for including nucleotide sequence, nucleotide sequence coded any TFPI of the invention The amino acid sequence of antibody and its antigen-binding fragment (includes but not limited in table 33 antibody or its antigen-binding fragment provided Amino acid sequence, such as SEQ ID NO：The amino acid sequence of 21-174), and with antibody binding of the present invention to identical epitope And/or the amino acid sequence of any antibody of competition binding TFPI.
On the other hand, the present invention provides the polynucleotides and its variation of coding TFPI antibody, wherein these variation multinuclears Thuja acid encode with any TFPI antibody amino acids sequences disclosed by the invention have at least 70%, at least 75%, at least 80%, extremely Few 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%th, the amino acid sequence of at least 96%, at least 97%, at least 98% or at least 99% sequence thereto.
In other embodiments, the nucleic acid and sheet of the variant polynucleotide sequence comprising coding TFPI antibody or part thereof Degree of relevancy between any specific nucleotide sequence of disclosure of the invention can be by testing the variant sequence thereof (or its complement) It is no to be measured with specific nucleotide sequence (or its complement) under moderate or high stringency in the RNA markings or the DNA markings Hybridize in form.Exemplary and nonrestrictive " moderate stringency " is included in 5 × SSC, 0.5%SDS, 1.0mM EDTA (pH8.0) prewashing in solution；At 50 DEG C to 65 DEG C, hybridize in 5 × SSC, overnight；Then again at 65 DEG C with containing 0.1%SDS 2X, 0.5X and 0.2X SSC respectively cleaning 20 minutes, carry out it is secondary.
Exemplary and nonrestrictive " high stringency " or " high stringency conditions " refer to following：(1) low ion is used Intensity and high temperature are for cleaning, such as at 50 DEG C, the dodecyl sulphate of the sodium citrate of 0.015 sodium chloride/0.0015/0.1% Sodium；(2) at 42 DEG C, denaturant, such as formamide (such as 50% (v/v) formamide) and 0.1% N are used in crossover process Seralbumin/0.1%Ficoll/0.1% polyvinyl pyrrolidone/50mM sodium phosphate buffer agents, pH 6.5, adds 750mM chlorine Change sodium, 75mM sodium citrates；Or (3) at 42 DEG C, using 50% formamide, 5 × SSC (0.75M NaCl, 0.075M citric acids Sodium), 50mM sodium phosphates (pH6.8), 0.1% sodium pyrophosphate, 5 × Denhardt solution, through Supersonic handle salmon sperm dna (50 μ G/ml), 0.1%SDS and 10% dextran sulfate, and 42 DEG C with 0.2 × SSC (sodium chloride/sodium citrate) and 55 DEG C with 50% formamide cleans, and is then cleaned at 55 DEG C with the high stringency lotion being made of 0.1 × SSC containing EDTA.This area skill Art personnel will be appreciated that to adjust temperature, ionic strength etc. how according to need to adapt to the factor such as probe length.This area is common Technical staff also will be familiar with for carry out RNA traces or southern blotting technique measure standard technique with detect nucleotide sequence variants with Degree of relevancy between the specific nucleotide sequence of the disclosure.
The present invention is also comprising the polynucleotides with any these sequences complementation.Polynucleotides can be single-stranded (coding or antisense) Or double-strand, and can be DNA (genome, cDNA or synthesis) or RNA molecule.RNA molecule includes hnRNA molecules, and (it contains interior Correspond to DNA molecular containing son and in a manner of man-to-man) and do not contain the mRNA molecules of introne.Extra coding or non-coding Sequence can, but not necessarily exist in the present invention polynucleotides in.
Alternatively, variation also can substantially with natural gene, or part thereof or its complement it is homologous.These polynucleotides become physical efficiency It is enough to hybridize under moderate stringency with encoding the natural DNA sequence (or complementary series) of natural antibody.
Those of ordinary skill in the art will be appreciated that the degeneracy due to genetic code, have many nucleotide sequences to compile Code any TFPI antibody disclosed in this invention or part thereof.Some in these polynucleotides may with it is provided by the present invention Any specific nucleotide sequence of TFPI antibody has rather low sequence thereto, but encodes same amino acid sequence.However, The polynucleotides being varied from by the difference of Codon Usage are that the present invention especially considers.
Chemical synthesis, recombination method or PCR can be used to obtain for the polynucleotides of the present invention.Chemical polynucleotides synthesis side Method is well known in the art, and need not be described in detail in the present invention.The present invention can be used to carry for those skilled in the art The sequence of confession and commercially available DNA synthesizer produce required DNA sequence dna.
In terms of polynucleotides are prepared using recombination method, suitable carry can will be inserted into comprising the polynucleotides of required sequence In body, then the carrier can be introduced into suitable host cell and be used to replicate and expand (as institute of the invention is discussed further ).Polynucleotides can be by any method Insertion Into Host Cell as known in the art.Cell can by directly absorbing, born of the same parents Gulp down effect, transfection, F- mating or electroporation and introduce exogenous polynucleotide to convert., can be by the more nucleosides of the external source once introduced Acid is kept in the cell by non-integrated vector (such as plasmid) or in the form of being integrated into host cell gene group.So amplification Polynucleotides can be isolated by method well known in the art from host cell.See, e.g. Sambrook et al.,1989。
Alternatively, PCR allows repetition DNA sequence.Round pcr is well known in the art, and is described in U.S. Patent No. 4, 683,195th, 4,800,159,4,754,065 and No. 4,683,202 and PCR:The Polymerase Chain In Reaction, Mullis et al.eds., Birkauswer Press, Boston, 1994.
RNA can be by being obtained in suitable carrier using separated DNA and be inserted into suitable host cell. When cellular replication and DNA are transcribed into RNA, the subsequent RNA can utilize well known to those skilled in the art method (such as Sambrook et al., cited in 1989 (being same as above)) be separated.
Suitable cloning vector can build according to standard technique or can be selected from this area in available a large amount of cloning vectors Select.Although selected cloning vector can change according to the host cell to be used, useful cloning vector usually has certainly I replicate ability, can possess for specific restriction enzyme single target and/or can carry available for selection containing should The gene of the mark of the clone of carrier.Suitable example includes plasmid and bacterial virus, such as pUC18, pUC19, Bluescript (such as pBS SK+) and its derivative, mp18, mp19, pBR322, pMB9, ColE1, PCR1, RP4, bacteriophage DNA and shuttle vector, such as PSA3 and pAT28.These and many other cloning vectors can from commercial suppliers such as BioRad, Stratagene and Invitrogen is obtained.
Further provide for expression vector.Expression vector is typically the reproducible multinuclear containing polynucleotides according to the present invention Thuja acid construct.It means that expression vector must be able in host cell or with episome (episome) or with chromosome The part form of DNA replicates.Suitable expression vector includes but not limited to plasmid, viral vector (including adenovirus, gland phase Close virus, retrovirus, sticking grain) and PCT Publication WO 87/04462 disclosed in expression vector.Carrier component is led to It often may include but be not limited to one or more following materials：Signal sequence；Replication orgin；One or more marker gene；It is suitable to turn Record control element (such as promoter, enhancer and terminator).In order to express and (translate), one or more translations are usually also required to Control element, such as ribosome bind site, translation initiation site and terminator codon.
Carrier and/or polynucleotides itself containing polynucleotides interested can pass through any of a variety of appropriate ways It is introduced into host cell, including electroporation, transfected using calcium chloride, rubidium chloride, calcium phosphate, DEAE- glucans or other materials； Particle bombardment (microprojectile bombardment)；Liposome transfection；And infection (such as wherein the carrier is infection Agent, such as vaccinia virus).The selection of introducing carrier or polynucleotides generally depends on the characteristic of the host cell.
The present invention also provides the host cell for including any polynucleotides described in the invention.Any energy overexpression is different The host cell of source DNA is used equally for the gene of separation coding antibody interested, polypeptide or protein.Mammalian hosts are thin The non-limiting examples of born of the same parents include but not limited to：Ape COS, people HeLa, human embryo kidney (HEK) (HEK) 293, Sp2.0 and Chinese hamster ovary (CHO) cell.Referring also to PCT Publication WO 87/04462.Suitable nonmammalian host cells include prokaryotes (such as Escherichia coli (E.coli) or bacillus subtilis (B.subtillis)) and yeast (such as saccharomyces cerevisiae (S.cerevisae), schizosaccharomyces pombe (S.pombe)；Or Kluyveromyces lactis (K.lactis)).Express TFPI antibody Or immune combination mensuration can be used to detect for the screening of the host cell of its antigen-binding portion thereof, for example, ELISA, FACS or other Measure known to the those of ordinary skill of field.
Therefore, antibody of the invention (or its antigen-binding fragment) can be used suitable host cell and be produced to recombinate.Compile The nucleic acid of the code antibody or its antigen-binding fragment can be cloned into expression vector, then can be introduced into addition to produce and be exempted from Host cell (such as Bacillus coli cells, yeast cells, insect cell, COS cells, Chinese hamster ovary celI or the myeloma of epidemic disease globulin Cell) in obtain synthesizing monoclonal antibody in recombinant host cell.Exemplary host cells include Chinese hamster ovary celI, HEK 293 And Sp2.0 cells.
Expression vector can be used for directly expressing TFPI antibody.It is familiar to the person skilled in the art how to apply expression vector to obtain To expressing foreign protein in vivo.See, e.g. U.S. Patent No. 6,436,908；6,413,942；And No. 6,376,471.Table Administration up to carrier includes topically or systemically applying, including inject, orally administer, the insertion of particle gun or conduit is applied and local Using.According to some non-limiting embodiments, which is to be applied directly to liver, skeletal muscle, marrow or other groups Knit.
Targeted delivery can also be used to contain the therapeutic combination of expression vector or subgenomic polynucleotides.It is receptor-mediated DNA delivery techniques are described in, such as Findeis et al., Trends Biotechnol., and 1993,11:202；Chiou et al.,Gene Therapeutics:Methods And Applications Of Direct Gene Transfer, J.A.Wolff,ed.,1994；Wu et al.,J.Biol.Chem.,1988,263:621；Wu et al., J.Biol.Chem.,1994,269:542；Zenke et al.,Proc.Natl.Acad.Sci.USA,1990,87:3655；Wu et al.,J.Biol.Chem.,1991,266:In 338.In gene therapy protocol, contain more nucleosides for local application The therapeutic combination of acid is applied with the scope of about 100ng to the DNA of about 200mg.It can also be used during gene therapy protocol dense Spend scope about 500ng to about 50mg, about 1 μ g to about 2mg, about 5 μ g to about 500 μ g, about 20 μ g to about 100 μ g DNA.Treatment Property polynucleotides and polypeptides can be used gene delivery supporting agent deliver.The gene delivery supporting agent can be viral or non-viral source (one As referring to Jolly, Cancer Gene Therapy, 1994,1:51；Kimura,Human Gene Therapy,1994,5: 845；Connelly,Human Gene Therapy,1995,1:185；And Kaplitt, Nature Genetics, 1994,6: 148).Endogenous mammalian promoter or allogeneic promoter can be used to induce for the expression of these coded sequences.The coded sequence Expression but composing type or adjusting.
The carrier based on virus for delivering desired polynucleotides and being expressed in desired cell is this area Well known.The exemplary supporting agent based on virus includes but not limited to recombinant retrovirus (see, e.g. PCT Publication the WO 90/07936；WO 94/03622；WO 93/25698；WO 93/25234；WO 93/11230；WO 93/10218；WO No. 91/02805；U.S. Patent No. 5,219,740 and No. 4,777,127；GB patents the 2,200,651st；And EP patents the 0th 345 No. 242), carrier based on α viruses (such as Sindbis viral vectors, Semliki forest virus (ATCC VR-67； ATCC VR-1247), Ross river (Ross River) virus (ATCC VR-373；ATCC VR-1246) and Venezuela's horse brain Scorching (Venezuelan equine encephalitis) virus (ATCC VR-923；ATCC VR-1250、ATCC VR 1249； ATCC VR-532)) and adeno-associated virus (AAV) carrier (see, e.g. PCT Publication WO 94/12649, WO 93/03769； WO 93/19191；WO 94/28938；WO 95/11984 and WO 95/00655).Also it can use and apply such as Curiel, Hum.Gene Ther.,1992,3:The DNA of the adenovirus of connection inactivation described in 147.
Also non-viral delivery supporting agent and method can be used, including but not limited to connects or be not connected with the adenopathy individually inactivated Poison polycationic condensation DNA (see, e.g. Curiel, Hum.Gene Ther., 1992,3:147)；Ligand connection DNA (see, e.g. Wu, J.Biol.Chem., 1989,264:16985)；Eukaryotic delivers supporting agent cell (see, e.g. U.S. State's patent the 5,814,482nd；PCT Publication WO 95/07994；WO 96/17072；WO 95/30763；With WO 97/ No. 42338) and nucleic acid charging neutrality or and cell membrane fusion.Also naked DNA can be used.Exemplary naked DNA introduction method description In PCT Publication WO 90/11092 and U.S. Patent No. 5,580,859.Can be as the liposome of gene delivery supporting agent It is described in U.S. Patent No. 5,422,120；PCT Publication WO 95/13796；WO 94/23697；WO 91/14445；And In EP 0524968.Extra method is described in Philip, Mol.Cell Biol., and 1994,14:2411 and Woffendin, Proc.Natl.Acad.Sci.,1994,91:In 1581.
The sequence of desired antibody (or its antigen-binding fragment) and encode these antibody (or its antigen-binding fragment) Standard sequence methods can be used to measure for nucleic acid.The nucleotide sequence for encoding desired antibody (or fragment) can be inserted into for recombinant production And in other carriers (such as clone and expression vector) of characterization.Heavy chain (or fragment of the heavy chain) and light chain (or the light chain Fragment) it can be cloned in same vehicle or different carriers.
Suitable clone and expression vector can include various components, such as promoter, enhancer and other transcriptional regulatories Sequence.The carrier also can allow constant region for immunoglobulin sequence to be moved between different carriers through construction.
Antibody fragment can by proteolysis or other antibody degradation methods, by recombination method or by chemical synthesis come Produce.The polypeptide of antibody, the shorter polypeptide of especially up to about 50 amino acid can be prepared conveniently by chemical synthesis.Chemistry Synthetic method is as is generally known in the art and commercially available.
Antibody disclosed by the invention or its antigen-binding fragment can be affine sexually matured.It is for example, affine sexually matured anti- Body can by method as known in the art come manufacture (Marks et al., 1992, Bio/Technology, 10:779-783； Barbas et al.,1994,Proc.Nat.Acad.Sci.USA 91:3809-3813；Schier et al.,1995, Gene,169:147-155；Yelton et al.,1995,J.Immunol.,155:1994-2004；Jackson et al., 1995,J.Immunol.,154(7):3310-3319；Hawkins et al.,1992,J.Mol.Biol.,226:889-896； And WO2004/058184).
4. formulation and purposes
Antibody described in the invention or antigen-binding fragment can be configured to pharmaceutical dosage form.With freeze-dried formulation or aqueous solution shape The pharmaceutical dosage form of formula can further include pharmaceutically acceptable carrier, excipient or stabilizer (Remington:The Science and practice of Pharmacy 20th Ed.,2000,Lippincott Williams and Wilkins,Ed.K.E.Hoover).Acceptable carrier, excipient or stabilizer are under the dosage and concentration to recipient It is nontoxic, and buffer can be included, such as phosphate, citrate and other organic acids；Antioxidant, including ascorbic acid And methionine；Preservative (such as hexadecyldimethylamine benzyl ammonium chloride；Hexamethonium chloride；Benzalkonium chloride, benzethonium chloride； Phenol, butyl or benzyl alcohol；Alkyl parabens class, such as methyl or propyl para-hydroxybenzoate；Catechol；Between Benzenediol；Cyclohexanol；3- amylalcohols；And metacresol)；Low molecular weight (less than about 10 residues) polypeptide；Protein, such as serum are white Albumen, gelatin or immunoglobulin；Hydrophilic polymer, such as polyvinyl pyrrolidone；Amino acid, such as glycine, paddy ammonia Acid amides, asparagine, histidine, arginine or lysine；Monosaccharide and disaccharide and other carbohydrate, including glucose, sweet dew Sugar or glucan；Chelating agent, such as EDTA；Carbohydrate, such as sucrose, mannitol, trehalose or sorbierite；Forming salt contend with from Son, such as sodium；Metal composite (such as Zn- protein complexes)；And/or nonionic surfactant, such as TWEENTM、 PLURONICSTMOr polyethylene glycol (PEG).Pharmaceutically acceptable excipient is further described in the present invention.
Antibody described in the invention or antigen-binding fragment can be used for various treatments or diagnostic purpose.Such as the antibody or Its antigen-binding fragment can be used as compatibility scarvenger (such as purification TFPI), as diagnosticum (such as examining Survey the TFPI expression in specific cells, tissue or serum).
The antibody of the present invention and the exemplary treatment purposes of antibody fragment include treatment thrombopenia (thrombocytopenia), blood platelet disorders (illness of platelet function or number) and hemorrhagic conditions (such as hemophilia A, Hemophilia B and congenital XI factor deficiency).The antibody and antibody fragment can also be used for the uncontrolled bleeding in treatment indication, such as create Wound and hemorrhagic stroke.The antibody and antibody fragment can also be used for prophylactic treatment (such as before surgical operation).
Especially, antibody described in the invention or antigen-binding fragment can be used for treatment blood coagulation shortage or clotting defect. For example, antibody described in the invention or antigen-binding fragment can be used for reducing or suppress TFPI and the interaction of FXa, or subtract Few TFPI dependences suppress TF/FVIIa/FXa activity.
In terms for the treatment of use, antibody described in the invention or antigen-binding fragment can be applied to the food in one's mouth by routine techniques Newborn animal, the especially mankind, such as pass through vein (with bolus (bolus) or passing through the continuous infusion within a period of time), muscle In interior, peritonaeum, brain keel, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, part or pass through suction.Antibody or antigen binding fragment Section also be adapted for by knurl, around tumour, intralesional or perilesional approach applies.
Therefore, on the one hand, the present invention provides the method for reducing tissue factor approach restrainer (TFPI) activity, it includes The object needed to there is this applies the antibody or antigen-binding fragment of the invention of therapeutically effective amount.On the other hand, it is of the invention The method for shortening the bleeding time is provided, it includes the antibody to there is the object that this is needed to be described using the present invention of therapeutically effective amount Or antigen-binding fragment.
In certain embodiments, which is the mankind.
In certain embodiments, which suffers from or easily occurs clotting defect.Clotting defect includes, such as vascular blood Friendly disease (von Willebrand disease) (vWD), Hemophilia A and B or C and other blood platelet disorders (such as congenital blood is small Board defect, congenital and the storage pool deficiency disease day after tomorrow, prolonged bleeding time).
In certain embodiments, the antibody or antigen-binding fragment that the present invention describes pass through subcutaneous administration.In some realities Apply in scheme, the antibody or antigen-binding fragment that the present invention describes pass through intravenous administration.
The pharmaceutical composition can be applied to the object of this needs with different frequency, and what which can be with bleeding episode is tight Principal characteristic and change, in preventative-therapeutic situation, change such as the seriousness of patient's clotting defect.
Said composition can be applied to patient in need with bolus or by continuous infusion.For example, bolus is applied with Fab pieces The antibody of Duan Chengxian, amount used can be 0.0025 to 100mg/kg (weight), 0.025 to 0.25mg/kg, 0.010 to 0.10mg/ Kg or 0.10 to 0.50mg/kg.In terms of continuous infusion, the amount of application of the antibody presented with Fab fragments can be small 1 to 24 When, 1 to 12 it is small when, 2 to 12 it is small when, 6 to 12 it is small when, 2 to 8 it is small when or 1 to 2 it is small when in a period of deliver 0.001 to 100mg/ Kg (weight)/minute, 0.0125 to 1.25mg/kg/ minute, 0.010 to 0.75mg/kg/ minute, 0.010 to 1.0mg/kg/ point Clock or 0.10 to 0.50mg/kg/ minute.
In terms of the antibody of full length antibody (the having complete constant region) form of administration, dosage can be about 1mg/kg to about 10mg/kg, about 2mg/kg are to about 10mg/kg, about 3mg/kg to about 10mg/kg, about 4mg/kg to about 10mg/kg, about 5mg/kg To about 10mg/kg, about 1mg/kg to about 20mg/kg, about 2mg/kg to about 20mg/kg, about 3mg/kg to about 20mg/kg, about 4mg/kg to about 20mg/kg, about 5mg/kg are to about 20mg/kg, about 1mg/kg or more, about about 2mg/kg or more, 3mg/kg Or more, about 4mg/kg or more, about 5mg/kg or more, about more than 6mg/kg, about 7mg/kg or more, about 8mg/kg or more More, about 9mg/kg or more, about 10mg/kg or more, about 11mg/kg or more, about 12mg/kg or more, about 13mg/kg or More, about 14mg/kg or more, about 15mg/kg or more, about 16mg/kg or more, about about 17mg/kg or more, 19mg/ Kg or more or about 20mg/kg or more.Frequency of administration is by the order of severity depending on the patient's condition.Frequency range can be three-times-weekly To every two weeks or once every three weeks.
In addition, said composition can be applied to patient by being subcutaneously injected.For example, can by dosage be 1 to 100mg anti-TFPI Antibody once a day, every 2 days once, every 3 days once, every 4 days once, every 5 days once, every 6 days once, weekly it is secondary, weekly, Every two weeks or monthly by be subcutaneously injected be applied to patient.
In certain embodiments, the pharmaceutical composition be by timetable weekly by subcutaneous administration about 0.1mg/kg extremely About 10mg/kg, about 0.5mg/kg to about 10mg/kg, about 1mg/kg to about 10mg/kg, about 1.5mg/kg to about 10mg/kg, about 2mg/kg to about 10mg/kg, about 0.1mg/kg are to about 8mg/kg, about 0.5mg/kg to about 8mg/kg, about 1mg/kg to about 8mg/ Kg, about 1.5mg/kg to about 8mg/kg, about 2mg/kg to about 8mg/kg, about 0.1mg/kg to about 5mg/kg, about 0.5mg/kg extremely About 5mg/kg, about 1mg/kg are to about 5mg/kg, about 1.5mg/kg to about 5mg/kg, about 2mg/kg to about 5mg/kg, about 0.5mg/ Kg, about 1.0mg/kg, about 1.5mg/kg, about 2.0mg/kg, about 2.5mg/kg, about 3.0mg/kg, about 3.5mg/kg, about 4.0mg/ Kg, about 4.5mg/kg, about 5.0mg/kg, about 5.5mg/kg, about 6.0mg/kg, about 6.5mg/kg, about 7.0mg/kg, about 7.5mg/ The dosage of kg, about 8.0mg/kg, about 8.5mg/kg, about 9.0mg/kg, about 9.5mg/kg or about 10.0mg/kg.
In certain embodiments, which is to pass through subcutaneous administration about 2.0mg/kg by timetable weekly Dosage.In certain embodiments, which is the agent by subcutaneous administration about 3.0mg/kg by timetable weekly Amount.
The antibody and antibody fragment that the present invention describes can be applied in combination with monotherapy or with other therapies to solve to stop blooding It is abnormal.For example, one or more antibody of the invention (or antibody fragment) and coagulant (such as factor VIIa, the factor is co-administered VIII, factors IX or tranexamic acid (tranexamic acid)) it is useful to treatment hemophilia.
In one embodiment, there is provided for treating clotting defect or shortening the method in bleeding time, it includes administration (a) antibody or antigen-binding fragment of the invention of the first amount, and the Factor IX or factors IX of (b) second amount.Optionally, no Factor Ⅴ II is co-administered.In another embodiment, there is provided for treating clotting defect or shortening the method in bleeding time, It includes the antibody or antigen-binding fragment of the invention of (a) first amount of administration, and the Factor IX or the factor of (b) second amount IX.Optionally, factor Ⅴ II is not co-administered.It will be appreciated by those skilled in the art that when treating clotting defect, shorten the bleeding time Alternatively referred to as shorten the clotting time.
The present invention also includes the antibody of the present invention (or antibody fragment) comprising therapeutically effective amount and Factor IX or factors IX Pharmaceutical composition, wherein said composition do not include factor Ⅴ II." factor Ⅴ II " includes factor Ⅴ II and factor VIIa.
Biological deposits
The representative substances of the present invention were preserved in American type culture collection on July 22nd, 2015 (American Type Culture Collection, 10801University Boulevard, Manassas, Va.20110-2209,USA).It is anti-that plasmid vector mAb-TFPI-106VH with ATCC preserving numbers PTA-122329 includes coding The DNA Insert Fragments of body TFPI-106 heavy chain variable regions, and the plasmid vector mAb- with ATCC preserving numbers PTA-122328 TFPI-106VL includes the DNA Insert Fragments of encoding antibody TFPI-106 light chain variable regions.The preservation is used for according to international recognition The microorganism of proprietary program preserves the regulation of budapest treaty (budapest treaty).This ensures the culture of the preservation from guarantor It can keep surviving within 30 years from Tibetan.The preserved material will be provided and by Pfizer according to the clause of budapest treaty by ATCC Agreement control between ATCC, this agreement ensure that the culture offspring of the preservation is when relevant United States Patent (USP) is issued or in office What U.S. or foreign patent application (be subject to first comer) it is open to the public when permanent and unrestricted availability, and ensure by U.S.Commissioner of Patents and Trademarks are according to 122 and the of 35U.S.C.Section Commissioner ' s rules (including 37C.F.R.Section 1.14, with particular reference to 886OG 638) decision offsprings' can The property used.
Dead when if the culture that present assignee has agreed to storage is cultivated under suitable conditions or loss or damage It is bad, another same substance will be replaced in time in notice.The availability of the material of preservation be understood not to allow run counter to appoint What government is according to the license for implementing the present invention under its Patent Law mandate.
Embodiment
The present invention is described in further detail by reference to following experiments embodiment.Unless otherwise prescribed, these are implemented The purpose that example is merely to illustrate, and be not intended for limiting.Therefore, the present invention should not be construed as limited to following in either side Embodiment, and should be interpreted to include becomes obvious any and all change because of teaching provided by the present invention.
Embodiment 1：Experiment material and method
1.TFPI protein reagents
Listed in table 1 for being immunized, phage display selection and the protein reagent for characterizing anti-TFPI antibody, and its sequence ID is listed in Table 2 below.
TFPI constructs (pSMED2 carriers) transient expression is set to be harvested in HEK293F cells and when 120 is small after transfection Conditioned medium.Albumen interested is captured from conditioned medium using nickel agarose HP (Nickel Sepharose HP) Matter is simultaneously further purified by size exclusion chromatography.From Haematologic Technologies, Inc. obtain factor Xa and Factor X.The chromogenic substrate of the Amidolytic measure for factor Xa is obtained from Sekisui DiagnosticsFXa。
Table 1
For being immunized, phage display selection and identify the protein reagent of anti-TFPI antibody
Table 1
TFPI reagent sequences identification number, description and sequence
2. antibody reagent
Monoclonal antibody (reference antibody 2A8,2A8-200,3F18, hz4F36) for comparative purposes is listed in Table 3 below.It is anti- Body description, source and sequence are listed in Table 4 below (Fig. 5).Light chain and sequence of heavy chain are cloned into in appropriate carrier simultaneously transient expression In human embryo kidney (HEK) -293 (HEK-293) cell, then pass through Protein A sepharose (Protein A Sepharose) and size exclusion Chromatography purifies.Mab2974 is obtained from R＆D systems (catalogue #MAB2974).
Table 3
Light chain (LC) CDR1,2,3, can lighten (VL), constant light (CL), light chain (LC), heavy chain (HC) CDR1,2,3, it is variable Weight (VH), constant heavy (CH) and heavy chain (HC) region.The sequence composition of No. SeqID is in table 4.
3.TFPI combinations ELISA
Utilize AviTagTMSystem will recombinate humTFPI K1K2, murTFPI K1K2, cynTFPI K1K2, ratTFPI K1K2, rabTFPI K1K2 or TFPI2 biotinylations, and with 1 × 10 in ELISA measures buffer-8The concentration of M is captured to On coated 96 orifice plate of Greiner streptavidins.The anti-TFPI antibody of purifying is diluted in ELISA measures buffer 1 μ g/ml, then serial dilution 3 is again to produce 8- point dilution series.The diluted antibody that volume is 100 μ L is added in each hole. By the plate be incubated at room temperature 2 it is small when.After cleaning the plate with PBS/0.05%Tween20, by the plate and with 1:10,000 dilutions The goat anti-mouse IgG-Fc polyclonal antibodies (Pierce) being conjugated with horseradish peroxidase together be incubated.Be incubated 1 it is small when Afterwards, the antibody of combination is detected by adding tmb substrate solution.Read the absorbance in 450nm and pass through GraphPad Prism software analysis datas.
4. surface plasma body resonant vibration (SPR)
Pass through amine coupling anti-human IgG antibody (catalog number BR-1008-39, GE Healthcare) to carboxy methylation Portugal All four flow cells on the coated sensor chip of glycan (CM5) (catalog number BR100530, GE Healthcare) (flow cell) prepares anti-human Fc sensor chips.By injecting 400mM 1- ethyls -3- (3- dimethyl aminopropyls) carbon The 1 of diimmonium salt hydrochlorate (EDC) and n-hydroxysuccinimide (NHS):1 mixture 7 minutes, was lived with 10 μ of flow velocity l/ minutes Change the flow cell.Anti- human IgG antibodies are diluted to 25 μ g/ml in 10mM sodium acetates (pH 5.0) and are divided with 10 μ l/ of flow velocity Clock is injected on all flow cells 7 minutes.All flow cells are closed 7 minutes with 1M monoethanolamines HCL (ETH), flow velocity divides for 10 μ l/ Clock.The final fixed level of the capture antibody is about 10,000 resonance units (RU).For fixed and dynamic (dynamical) runtime buffer Liquid is 10mM HEPES pH 7.4,150mM NaCl, 3mM EDTA, 0.05% (v/v) Tween-20 (HBS-EP+).For table The combination of anti-TFPI antibody and mankind TFPI is levied, which is diluted to 0.5 μ g/ml in HBS-EP+, and by being fixed on circulation Anti-human IgG in pond 2,3 and 4, captured 30 seconds to 1 minute with the 5 μ flow velocitys of L/ minutes, to reach 70 to 300RU capture water It is flat.Using flow cell 1 as referring to surface.After capturing antibody, flow velocity is increased into 50 μ L/ minutes and by buffer or in HBS- The mankind TFPI that concentration range is 0.2nM to 200nM in EP+ is injected on all flow cells to combine 1.0 minutes, then allows it Dissociation 10 to 15 minutes.Using the buffer of collected each capture antibody circulate for it is dual with reference to (Myszka, D.G.J.Mol.Recognit.12,279-284(1999)).In each end cycle, pass through 60 pulse per second (PPS) 3M MgCl2Make Whole anti-igg surface regeneration.At 25 DEG C, with the collection rate of 10Hz on BIAcore T200 instruments (GE Healthcare) Carry out kinetic determination.By the way that data are fitted to the 1 of BIAcore T200 assessment software versions 1.0 (GE)：1 model measures Speed constant and compatibility.
5. factor XaTFPI suppresses to recover measure
For evaluating in vitro in the presence of the TFPI of inhibition concentration, the anti-TFPI antibody of purifying recovers the energy of factor Xa activity Power.By in PBS diluted concentration range be 1nM to 500nM anti-TFPI antibody with activity buffer agent (20mM HEPES, pH8.0、150mM NaCl、5mM CaCl2, 0.5mg/mL BSA) in 10nM recombinant human TFPI K1K2 or 10nM rabbits TFPI K1K2 albumen preincubate 30 minutes at 37 DEG C.Add factor Xas of the 2nM derived from human plasma and by reactant 37 It is incubated 30 minutes at DEG C.Chromogenic substrate Spectrozyme Xa are added thereto, make 100 μ l end reaction volume it is final dense Spend for 500 μM.Control reaction is included without factor Xa to control measure background, without TFPI to allow the generation of FXa most (100% activity) or the reaction without anti-TFPI antibody (single PBS).Immediately in SpectraMax M5e multi-mode disc types On analyzer, in a period of 60 minutes, the absorbance of reaction is read at 405nm wavelength at intervals of two minutes.With Prism Graph Pad softwares calculate EC50。
6. two benches TF-FVIIa-FX suppresses to recover measure
Evaluating in vitro in the presence of the TFPI of inhibition concentration, the anti-TFPI antibody of purifying recover factor VIIa-tissue because The ability of sub- activity.The anti-TFPI antibody that concentration range is 1nM to 500nM is recombinated into TFPI with the 10nM in activity buffer agent K1K2 albumen preincubate 30 minutes at 37 DEG C.By tissue factor esterified about 1pM and 1nM recombinant factors VIIa (NovoSeven) add in the reactant and be incubated 5 minutes at 37 DEG C.The human Factor X of 150nM is introduced into the reactant. Chromogenic substrate Spectrozyme Xa are added in each hole, the ultimate density for making the end reaction volume of 100 μ l is 500 μM. Control reaction is included without factor VIIa, without tissue factor, without factor X, without TFPI or without anti-TFPI antibody (individually PBS) reaction.Immediately on SpectraMax M5e multi-mode disc type analyzers, in a period of 60 minutes, every 2 points Clock reads the absorbance of reaction at 405nm wavelength.EC is calculated with Prism Graph Pad softwares50。
7. fibrin ferment generation experiment (TGA)
The anti-TFPI of purifying is assessed in fibrin ferment generation experiment using automatic fibrin ferment generation figure (CAT) system of calibration Antibody recovers the ability of fibrin ferment generation in the blood plasma that Factor VIU activity weakens.Will in PBS diluted concentration for 1nm extremely The anti-TFPI antibody of 500nm, which is introduced, to be lacked blood plasma and the low reagents of PPP- containing human Factor VIII, contains 4 μM of phosphatide and 1pM groups In the reactant for knitting the factor.Control reactant uses PBS, without antibody.Addition contains fluorometric thrombin substrate and CaCl2's Fluca buffers react to trigger.Fluoroskan Ascent disc type analyzers are used immediately, use Thrombinoscope Software is in 60 minutes, every the fluorescence that 20 seconds read each reactant.By each reactant with containing PBS, fibrin ferment calibrator, because Sub- VIII lacks blood plasma and the calibrator control wells of FLUCA buffers compare.Use Thrombinoscope softwares (Thrombinoscope BV versions) analysis Thrombinoscope fibrin ferments formation curve (nM fibrin ferments are to the time) is with deduction Go out time delay, crest height and reach time to peak and area under the curve or intrinsic coagulation enzyme potential (ETP).Use these Data carry out calculating speed index (peak value concentration of thrombin/reach time to peak-time delay).
Also the anti-TFPI antibody for assessing purifying recovers fibrin ferment generation in the rabbit blood plasma that Factor VIU activity weakens Ability.At 37 DEG C, control that the anti-FVIII antibody (GM-8015) or concentration using ultimate density as 100 μ g/mL are 100 μ g/mL Mouse anti human class IgG2a handles normal New Zealand White Rabbit blood plasma 60 minutes.Adding reacting hole not long ago, rabbit blood plasma was being existed With 1 in buffer (20mM HEPES, 140mM NaCl):3 dilutions.As described above, coagulated with the FVIII rabbit blood plasma neutralized Hemase generation experiment.
8. produce machin (cynomolgus) TFPI K2 domains for structural research
Machin (cyno) TFPI K1K2 (table 1) expression is harvested into bar in HEK293 cells and when 120 is small after transfection Part culture medium.By the Cyno TFPI K1K2120 of purifying and human neutrophil elastoser (HNE) with 1:70 (rub You compare HNE:TFPI) 120 minutes are incubated together in room temperature to crack.Anion exchange color is utilized on HQ50 (Poros) Spectrometry separates cyno K1 from cyno K2.Size exclusion spectrum, which is carried out, using Superdex 75 is used as final purification step.Make The C-terminal of residue A viTag from cyno K2 domains is trimmed with interior protease A spN.
9. produce monoclonal antibody/cyno TFPI K2 compounds for structural research
According to the scheme of manufacturer (Thermo/Pierce), with the fixed anti-TFPI antibody of papain digestion 4D8.b1, TFPI-23, TFPI-24,2A8-200 (table 3) and Mab 2974 (R＆D systems).Purified using MabSelect SuRe Carry out the Fab of self-digestion, then itself and cyno TFPI K2 are formed into compound.Then, it is Fab/cyno TFPI K2 compounds is dense It is reduced to about 16mg/ml.Reuse the concentrate screening protein crystallization condition.
Embodiment 2. produces the anti-TFPI antibody of mouse
1. mouse immune and generation hybridoma
By a cohort, the BALB/c mouse of totally 5 is each used in the 5 μ g humTFPI that emulsify in complete Freund's adjuvant K1K2 and 5 μ g murTFPI K1K2 protein mixture subcutaneous inoculations.Then weekly with incomplete Freund's adjuvant emulsify or Diluted protein mixture is by mouse immune 2 times in PBS.It is (immune at the 5th and the 7th time respectively at the 17th day and 27 days Blood sample is taken afterwards), by the presence that anti-TFPI antibody is circulated in ELISA test seras.At the 27th day, every mouse was led to Cross intraperitoneal injection and receive last time protein mixture (10 μ g) booster shots.After four days, harvest draining lymph node (armpit, Groin is Ji popliteal) and by the lymph node cells collected and P3X63.Ag8.653 cells with 1：1 ratio is mixed and carried out electric-thin Born of the same parents are merged.The plating cells of fusion are being supplemented with FBS (25%), NCTC-109 (12.5%), Glutamax (1%), mould Element-streptomysin (1%), hybridoma clone replenishers (5%) and HAT (1 × 10-4M hypoxanthine, 4 × 10-7M aminopterin-induced syndromes (aminopterin) and 1.6 × 10-5M thymidines) RPMI1640 culture mediums in.The fusion is tested after 14 days by ELISA to hybridize The combination of knurl culture supernatant and humTFPI K1K2.Activity and its effect in functional examination are combined according to it, selection comes It is used to further characterize from the antibody of three hybridomas 4D8,6B7 and 7A4.
2. clone and the sequencing of the anti-TFPI antibody derived from hybridoma
Prepare the RNA from hybridoma 4D8,6B7 and 7A4 and cloned by RT-PCR and obtain the antibody from the expression Variable region DNA sequence dna.PCR product is cloned into TOPO-TA cloning vectors, is then sequenced by conventional method.From hybridoma 6B7 and 7A4 detects a heavy chain and light chain cdna pair.Two heavy chains and light chain cdna are detected from 4D8.
Embodiment 3. characterizes the anti-TFPI antibody of Mouse Hybridoma Cells
Parental hybridoma 4D8,6B7 and 7A4 are subcloned to obtain monoclonal hybridoma system by limiting dilution.It is logical Cross ELISA and screen the reactivity with humTFPI K1K2 to identify positive subclone and expand.By one from each hybridoma The antibody further characterization of the purifying of subclone.
1.TFPI is combined
Pass through anti-TFPI antibody 4D8.B1,6B7.C5 and 7A4.D9 and recombined human of protein combination ELISA test purifying The combination of class and rabbit TFPI albumen.Each antibody is shown in the EC50 values of humTFPIK1K2-aviHis10 and rabTFPI K1K2 In table 5.
Table 5
EC50 (nM) value of the anti-TFPI monoclonal antibodies of mouse to the mankind and rabbit TFPI
Antibody EC50(nM)humTFPI K1K2 EC50(nM)rabTFPI K1K2
4D8.B1 0.0959 0.0976
6B7.C5 0.1209 0.1289
7A4.D9 0.0887 0.0887
Surface plasma resonance laboratory is carried out to assess the anti-TFPI antibody on human class of the mouse of the purifying and rabbit TFPI The compatibility of K1K2 albumen.The k that each antibody is combined with the mankind and rabbit TFPI K1K2a、kdAnd KDValue is shown in table 6.
Table 6
Kinetic measurement of the anti-TFPI Mouse Hybridoma Cells clone with reference to the mankind and rabbit TFPI
Analyte Ligand ka(1/Ms) kd(1/s) KD(nM)
humTFPI K1K2 4D8.B1 9.58x 105 1.14x 10-3 1.19
humTFPI K1K2 6B7.C5 5.52x 105 3.63x 10-3 6.58
humTFPI K1K2 7A4.D9 1.48x 106 9.21x 10-3 6.22
rabTFPI K1K2 4D8.B1 2.01x 106 1.26x 10-3 0.63
rabTFPI K1K2 6B7.C5 1.14x 106 5.64x 10-3 4.95
rabTFPI K1K2 7A4.D9 5.30x 106 1.88x 10-2 3.55
2. active determination in vitro
Suppress to test anti-TFPI mouse in recovery measure and fibrin ferment generation experiment (TGA) in FXa and TF-FXa-FVIIa The activity of monoclonal antibody.Most effective antibody 4D8.B1 is selected further to be studied.
Table 7
Anti- TFPI mouse monoclonal antibodies suppress to recover measure and fibrin ferment generation experiment in FXa and TF-FXa-FVIIa (TGA) activity in
Antibody FXa EC50(nM) FXa-FVIIa EC50(nM) Rate Index of the TGA in 20nM
4D8.B1 5.9 6.67 26.3
6B7.C5 13.9 12.13 23.5
7A4.D9 22.3 9.35 23.3
Embodiment 4. produces chimeric and humanized antibody from clone 4D8
1. produce mice human's Chimeric antibodies 4D8
Variable region cDNA derived from hybridoma 4D8 is subcloned into mammalian expression vector to produce wherein mouse Heavy chain variable region is merged with 1 3M of human IgG (SEQ 20, table 4) in framework, and mouse light chain variable region and mankind Ig κ are constant The chimeric antibody that area's (SEQ 62, table 4) is merged in framework.The chimeric constructs are transiently transfected into HEK293 cells.With All possible heavy and light chain combination carries out the transient transfection of totally four times to identify the correct weight and light chain from hybridoma 4D8 It is right.Claimed to be hu-mu 4D8 chimeras (table 3 and 4) from the antibody for wherein once transfecting generation.
2. characterize mouse-human chimeric's antibody 4D8 (hu-mu 4D8)
Mu-hu 4D8 chimeras and the mankind and rabbit TFPI are tested by protein combination ELISA (table 8) and SPR (table 9) The protein bound abilities of K1K2.The numerical value that the mouse Mab 4D8.B1 that the KD and EC50 values are purified with those are measured is very suitable, The transplanting for the mouse variable region for proving to be transplanted to 1 background of human IgG can retain with reference to activity.
Table 8
EC50 (nM) value of mu-hu 4D8 chimeras to the mankind and rabbit TFPI
Antibody EC50(nM)humTFPI K1K2 EC50(nM)rabTFPI K1K2
mu-hu 4D8chimera 0.0577 0.0680
Table 9
Kinetic measurement of the mu-hu 4D8 chimeras to the mankind and rabbit TFPI
Analyte Ligand ka(1/Ms) kd(1/s) KD(nM)
humTFPI K1K2 mu-hu 4D8chimera 9.58x 105 1.14x 10-3 1.19
rabTFPI K1K2 mu-hu 4D8chimera 2.01x 106 1.26x 10-3 0.63
3. humanization hu-mu 4D8 chimeras
It is transplanted to hu-mu 4D8 chimera sequence humanizations by CDR on human receptor's frame sequence.Select DP54 Framework and DPK9 frameworks.Then the combination of expression weight and light chain construct (referring to table 3).Tested in ELISA combination mensurations anti- Body is combined (table 10) with the mankind and rabbit TFPI and test antibody is combined (table 11) with mankind TFPI in SPR combination mensurations.
Table 10
EC50 (nM) value of humanization 4D8 antibody binds humans and rabbit TFPI K1K2 albumen
Table 11
The SPR analyses of humanization 4D8 antibody binding humTFPI K1K2 albumen
Antibody ka(1/Ms) Kd(1/s) KD,nM
4D8Vk 1.0x VH 1.0 6.77x 104 4.70x 10-4 6.94x 10-9
4D8Vk 1.0x VH 1.1 6.06x 104 1.54x 10-3 2.54x 10-8
4D8Vk 1.0x VH 1.2 2.38x 105 1.65x 10-4 6.95x 10-10
4D8Vk 1.0x VH 1.3 7.95x 104 5.71x 10-4 7.19x 10-9
4D8Vk 1.0x VH 1.4 7.55x 104 8.35x 10-4 1.11x 10-8
4D8Vk 1.1x VH 1.0 1.25x 105 8.35x 10-4 5.50x 10-10
4D8Vk 1.1x VH 1.1 1.93x 105 1.61x 10-4 8.32x 10-10
4D8Vk 1.1x VH 1.2 1.51x 105 9.49x 10-5 6.27x 10-10
4D8Vk 1.1x VH 1.3 1.59x 105 1.31x 10-4 8.23x 10-10
4D8Vk 1.1x VH 1.4 2.21x 105 5.28x 10-5 2.93x 10-10
Based on these data, 4D8Vk 1.1xVH 1.4 are selected to be further characterized and be named as hz4D8 (table 3). Suppress to recover to compare the anti-TFPI antibody of humanization in measure, two benches TF-FVIIa-FX suppression measure and fibrin ferment generation experiment (hz4D8) with the activity of mouse 4D8.B1FXa.Data in table 12 show that compared with mouse antibodies humanized antibody is in institute There is the activity that raising is respectively provided with three measure, this points out that TFPI is fully retained in humanized antibody with reference to activity.
Table 12
4D8.B1 and hz4D8 antibody suppresses to recover measure and fibrin ferment generation experiment (TGA) in FXa and TF-FXa-FVIIa In expression activitiy
Antibody FXa EC50(nM) FXa-FVIIa EC50(nM) Rate Index of the TGA in 20nM
4D8.B1 4.15 3.5 13.5
Hz4D8 1.87 1.57 15.7
Embodiment 5. produces extra anti-TFPI antibody by display technique of bacteriophage
1. anti-TFPI antibody is selected by phage display
4 wheel selections are carried out using the phage display library of the scFv antibody fragments derived from non-immunized non-human donor Single-chain fragment variable (scFv) antibody of the identification with reference to recombinant human and mouse TFPI K1K2 afterwards.It is small using streptavidin Pearl carries out phage selection in the solution.By triethanolamine (TEA) pH 11.5 or the MES pH 5.5 of 50mM with 140mM, 10 minutes bacteriophages for carrying out elution of bound are incubated on the gyrate shaker of room temperature, and neutralized with 1M Tris-HCl pH7.5.
The Escherichia coli ER2738 culture (phases of mid-term logarithmic phase have been grown to using the phage library infection 10mL of elution When in OD600It is about 0.5).At 37 DEG C, with phage-infect bacterium 30 minutes, do not shake, by centrifugal concentrating and carry out bed board, Grown overnight at 30 DEG C again.In next round selection, OD is reached by the 2 × TYAG/ tetracyclines for being seeded in 25mL600About 0.1, then make it grow to OD at 37 DEG C600Bacteriophage is saved for 0.3 to 0.5.With 1:The ratio of 20 cells/helper phage Do not shake incubation 30 minutes with MK13K07 helper phage superinfection cells and at 37 DEG C, then shaken 60 minutes in 150rpm.So Cell is centrifuged afterwards and precipitation is resuspended in the culture medium containing kanamycins/non-glucose.This culture is made at 25 DEG C Growth is overnight.The bacteriophage in supernatant is harvested after centrifugation and for the selection of next round.
2. prepare the thick all materials for being used for ELISA measure
According to used growth conditions, ScFv antibody fragments can be expressed on phage particle surface or in bacteria periplasm In the solution in space.In order to induce release scFv antibody fragments to enter in pericentral siphon, the Glycerol stocks from defrosting are seeded in Containing 2X TY culture mediums and 0.1% glucose/100 μ g/mL ampicillins 96 deep-well plates in, it is and small in 37 DEG C of growths about 4 When.The content (peripreps) of bacteria periplasm is discharged by osmotic shock.Plate is centrifuged and harvests the supernatant containing scFv Liquid.
Combination of the 3.ELISA measurement tables up to scFv and the mankind and mouse TFPI K1K2 in pericentral siphon
Choose 1984 clones altogether in being taken turns at random from the 2nd, 3 and 4 of all branching selections.Pass through periplasmic preps (periprep) TFPI scFv combinations strains (binder) are identified with reference to ELISA.By the biology that concentration is 1 μ g/mL in PBS The mankind and mouse TFPI K1K2 of elementization are coated on 384 hole NuncMaxisorp streptavidin plates.By TFPI K1K2 Solution removes and by the plate when room temperature is small with 0.05%Tween 20/1%BSA/PBS closings 1.Prepare periplasmic preps and When room temperature uses 6% isometric breast/1%BSA closings 1 small.The pericentral siphon scFv of the closing in 20 μ l/ holes and control antibodies are transferred to In appropriate plate and when incubation at room temperature 1 is small.Add with 1：2,000 diluted anti-myc horseradish peroxidases (HRP) or with 1:The scFv or anti-TFPI control antibodies combined with detection of 10,000 diluted Goat anti-Human's class HRP secondary antibodies.Using 3, 3', 5,5'- tetramethyl benzidine develop signal and are read on Envision disc types analyzer (Perkin Elmer) in 450nm Absorbance.Share 883 scFV clones and be accredited as TFPI combination strains.By 883 TFPI combinations scFv be sequenced with Identify unique clone.288 unique clones are selected to test TFPI/FXa competitive bindings.
4.ELISA identifies the scFv with the FXa competition bindings mankind and mouse TFPI K1K2
Test 288 unique clones altogether in FXa/TFPI competitive binding ELISAs.To concentration be 1 μ g/ in PBS The mankind FXa of mL is coated on 384 hole Nunc Maxisorp plates overnight.FXa solution is removed and uses the plate in room temperature When 0.05%Tween 20/1%BSA/PBS closings 1 are small.Prepare periplasmic preps and at room temperature with 6% breast/1% of volume When BSA closings 1 are small.The pericentral siphon scFv of the closing in 20 μ l/ holes and control antibodies are mixed with biotinylated humTFPI K1K2 And when incubation at room temperature 1 is small.Mixture is transferred to the coated plates of FXa and when incubation at room temperature 1 is small.Add with 1：2000 dilutions Streptavidin horseradish peroxidase to detect the TFPI of combination.Use 3,3', 5,5'- tetramethyl benzidines development letter Number, on Envision disc types analyzer (Perkin Elmer), read the absorbance in 450nm.Share 48 scFV antibody It is classified as the competitive inhibitor of TFPI/FXa combinations.
5.ScFv changes into human IgG
48 are selected altogether to show with reference to TFPI and inhibitory action has unique sequence in TFPI/FXa competitive ELISAs The scFV antibody of row, to be subcloned into human IgG -3M cloning vectors.Briefly, standard PCR amplification fragment is passed through.By VH Or VL fragments carry out gel-purified and connect into the mammal containing human IgG 1-3M (VH) or κ or λ constant regions (VK/VL) In expression vector.Then, the expression vector matched using VH and VK/VL is in 293 cells of HEK for instantaneous mammal table Reach and purify.
6. characterize the anti-TFPI antibody of human IgG -3M
48 kinds of anti-TFPI antibody are classified in various measure (including FXa and TF/FVII/FXa suppresses to recover measure). TFPI-3, TFPI-21, TFPI-23, TFPI-24 and TFPI-26 have desired property, such as TFPI is across species, with The combination power of humTFPI2K1K2K3 is low or without (table 13).The FXa and TF/FVIIa/FXa of this identical 5 kinds of antibody suppress to recover to survey Fixed number evidence is shown in table 14, and SPR combination data are shown in table 15.
Table 13
The display of TFPI antibody combines a variety of TFPI species (people, monkey, mouse, rabbit and rat) and is tied with mankind TFPI2 Close weak or nothing
Table 14
Activity of the TFPI antibody in FXa, TF/FVIIa/FXa suppress to recover measure and fibrin ferment generation experiment
Antibody FXa EC50(nM) FXa-FVIIa EC50(nM) TGA Velocity Index at 20nM
TFPI-3 207.9 37.4 NT
TFPI-21 80.64 54.1 24.71
TFPI-23 46 32.2 23.28
TFPI-24 23 9.3 23.21
TFPI-26 12.8 9.6 18.65
Table 15
The SPR binding kinetics of TFPI antibody on human class and mouse TFPI
Analyte Ligand ka(1/Ms) kd(1/s) KD(nM)
humTFPI K1K2 TFPI-3 6.25x 104 1.7x 10-3 27.05
humTFPI K1K2 TFPI-21 7.85x 105 3.47x 10-2 43.95
humTFPI K1K2 TFPI-23 9.18x 105 9.71x 10-3 9.89
humTFPI K1K2 TFPI-24 1.59x 105 8.62x 10-4 5.46
humTFPI K1K2 TFPI-26 2.05x 105 1.18x 10-3 5.79
murTFPI K1K2 TFPI-3 2.15x 105 8.24x 10-4 3.81
murTFPI K1K2 TFPI-21 1.73x 106 7.71x 10-3 4.54
murTFPI K1K2 TFPI-23 1.69x 106 2.48x 10-3 1.46
murTFPI K1K2 TFPI-24 2.48x 105 5.96x 10-3 24.1
murTFPI K1K2 TFPI-26 1.29x 106 4.76x 10-3 3.68
Embodiment 6. is mapped by the anti-TFPI antibody epitopes of SPR
Using sandwich SPR measure come to herein find and disclosed anti-TFPI antibody (TFPI-21, TFPI-23, TFPI-24,4D8,6B7.c5 and 7A4.D9) epitope mapping.Other known reference antibody (hz4F36,2A8-200 and Mab2974) it is also included within Epitope mapping experiments.Antibody 1 is fixed on CM5biacore chips using NHS chemistry.First Mankind TFPI (humTFPI K1K2) is injected on the chip until combining close to apparent equilibrium.After stopping injection mankind TFPI Antibody 2 is injected on the chip immediately.If antibody 2 combine the CM5 chip surfaces on antibody 1 and mankind TFPI it is compound Thing, then antibody 2 there is different and non-overlapped TFPI combinations epitopes (being denoted as+) relative to antibody 1.If antibody 2 shows no knot Close, then it is designated as significantly overlapping epitope (negative (-)) relative to antibody 1.If antibody 2 is shown with reference to weak, antibody 1 and 2 It is considered having in TFPI epitopes and partly overlaps and (be denoted as +/-).As shown in table 16, TFPI-21 has similar with TFPI-23 Epitope and the data display that TFPI-21 and TFPI-23 has the epitope entirely different with mab2974 and hz4F36.
Table 16
Use the epitope mapping of the anti-TFPI antibody of SPR sandwich determinations
Antibody 1 is fixed on the surface of CM5 chips.Then mankind TFPI (humTFPI K1K2) is expelled to surface On, until the measurement is close to apparent equilibrium.Stop TFPI injection after immediately injection of antibodies 2 with measure with antibody 1/TFPI compounds Combination."+" mark is given in the pairing of antibody 1 and 2 with entirely different epitope.Antibody with strong overlapping epitope is matched somebody with somebody To giving "-" mark.If antibody 2 is shown with reference to weak, antibody 1 and 2 is considered having in TFPI epitopes some parts overlapping (being denoted as +/-).
The human framework of 7. system genitale TFPI-23 antibody of embodiment
The variation of two kinds of TFPI-23 is prepared to increase the content of human framework's system genitale residue.TFPI-106 contains H1Q extremely The mutation (Kabat numberings) of E and H5V to L, and the mutation of TFPI-107 (table 3 and 4) containing H1Q to E, H5V to L and H94I to K (Kabat numberings).TFPI-106, TFPI-107 and TFPI-23 are expressed, after purification by SPR tests with humTFPI K1K2's With reference to.When data in table 17 are shown compared with TFPI-23 parental antibodies, TFPI-106 system genitales variation, which retains, all to be combined Compatibility.
Table 17
The SPR binding kinetics of TFPI-23 human framework's system genitale variations and mankind TFPI
When compared with parent's TFPI-23 antibody, TFPI-106 is shown to be improved with reference to appropriateness.
The human framework of 8. system genitale TFPI-24 antibody of embodiment
Prepare four kinds of TFPI-24VL variations (TFPI-110, TFPI-111, TFPI-112, TFPI-113) and and TFPI- 24VH sequences are matched.Prepare three kinds of TFPI-24VH variations (TFPI-108, TFPI-109, TFPI-114) and with TFPI-24VL sequences Row pairing.It is according to these data, optimal VL variations TFPI-113 and optimal VH variations TFPI-108 pairings is anti-to produce Body TFPI-118.By the combination of SPR tests TFPI-118 and TFPI-24 and mankind TFPI, the results show in table 18 goes out phase When binding kinetics.
Table 18
Compare the binding kinetics of TFPI-24 and human framework's variation TFPI-118 and mankind TFPI using SPR
Analyte Ligand ka(1/Ms) kd(1/s) KD(nM)
humTFPI K1K2 TFPI-24 9.18x 105 9.71x 10-3 3.68
humTFPI K1K2 TFPI-118 1.25x 105 1.19x 10-3 9.61
TFPI-118 shows the binding kinetics suitable with parent's TFPI-23 antibody.
The SPR binding kinetics of the anti-TFPI antibody of embodiment 9. and the TFPI from different plant species
Anti- TFPI antibody (TFPI-106, TFPI-118 and hz4F36) is analyzed by SPR to measure to from different animals TFPI (mankind (huTFPI K1K2), machin (cynTFPI K1K2), rabbit (rabTFPI K1K2), the mouse of species (murTFPI K1K2) and rat (ratTFPI K1K2)；Table 1) binding kinetics.Also compare in this experiment including three kinds (comparitor) antibody (hz4F36,2A8 and 2A8-200).
Table 19
Anti- TFPI antibody on human class, machin, rabbit, the SPR binding kinetics (K of rat and mouse TFPIdValue is two The average of experiment)
The anti-TFPI antibody/TFPI composite structures of embodiment 10.
1.4D8.b1Fab/cyno TFPI K2 composite structures
By 4D8.b1Fab and cyno TFPI K2 with 1：1 mixed in molar ratio is to form compound.Use Superdex 200 columns carry out last purifying.Compound is condensed into 12.6mg/ml for structural research.In 100mM Tris-HCl The crystal of TFPI K2+4D8Fab compounds is obtained in pH8.5-20%PEG10000.It produces diffraction and reachesBar-shaped crystalline substance Body.Synchrotron Data Collection is remotely performed by the instantaneous low-temperature protection of crystal and in Advanced Photon Source.Make Imaged frame is handled with software AutoPROC (Global PhasingLtd).The data belong to space group P212121, and structure cell is as follows：α=β=γ=90 °, each asymmetry unit have one again Compound.Use the homology model of 4D8Fab and structure (the RSCBProtein Data for the TFPI K2 domains that can openly obtain Bank；PDBcodes 1TFX and 4DTG) molecular replacement search is carried out to produce the parsing of compellent each component.Using soft Part autoBUSTER (Global PhasingLtd) carries out refine,The final R/Rfree factors be 0.1707 respectively With 0.2424, the RMSD of key isThe RMSD of angle is 1.26 °.Residue based on Fab/TFPI K2 interfaces it is hidden The surface region (BSA) and BSA percentages (%BSA) of Tibetan, measure the epitope and paratope of 4D8Fab.The K2 of following TFPI Residue in domain participates in directly contacting 4D8Fab (according to the epitope of BSA)：E101、P103、Y109、I110、T111、 Y113, F114, S119, Q121, C122, E123, R124, F125, K126 and L140.Residue bag in the heavy chain of following 4D8Fab Paratope containing heavy chain：D50, T57, L58, Y59, Q61, K64, D98, Y99 and D100.It is residual in the light chain of following 4D8Fab Base includes Light Chain Antigen paratope：H30, W50, H91, Y92, T93, T94, P95 and Y96.The epitope and paratope residue BSA and %BSA values be shown in table 20.
Table 20
As 4D8.b1Fab/cyno TFPI K2 composite structures median surface residue hiding surface region (BSA) and Anti- TFPI antibody 4D8.b1 epitopes and paratope residue defined in BSA percentages (%BSA)
(antibody light chain (LC) and heavy chain (HC) residue use Kabat definition numbering) is blockedOr the BSA of bigger, or Participate in electrostatic interaction) it is applied to BSA analyses
Residue Chain Residue # Classification BSA %BSA
GLU TFPIK2 101 Epitope 33.93 48.73
PRO TFPIK2 103 Epitope 34.91 74.82
TYR TFPIK2 109 Epitope 71.54 55.15
THR TFPIK2 111 Epitope 41.72 66.65
SER TFPIK2 119 Epitope 32.89 65.61
GLN TFPIK2 121 Epitope 67.99 86.51
GLU TFPIK2 123 Epitope 63.53 99.15
ARG TFPIK2 124 Epitope 147.09 97.31
LYS TFPIK2 126 Epitope 114.72 95.85
LEU TFPIK2 140 Epitope 41.62 60.14
ASP 4D8.b1HC 50 Paratope 5.98 52.56
THR 4D8.b1HC 57 Paratope 26.48 42.04
LEU 4D8.b1HC 58 Paratope 72.95 93.41
TYR 4D8.b1HC 59 Paratope 15.75 33.75
GLN 4D8.b1HC 61 Paratope 59.14 45.41
ASP 4D8.b1HC 98 Paratope 62.87 50.71
TYR 4D8.b1HC 99 Paratope 44.28 59.06
ASP 4D8.b1HC 100 Paratope 5.83 27.84
HIS 4D8.b1LC 30 Paratope 53.18 56.60
TRP 4D8.b1LC 50 Paratope 53.41 51.52
TYR 4D8.b1LC 92 Paratope 97.99 96.61
THR 4D8.b1LC 93 Paratope 39.89 73.42
THR 4D8.b1LC 94 Paratope 48.00 95.74
TYR 4D8.b1LC 96 Paratope 15.49 72.23
2.2A8 and 2A8-200Fab/cyno K1K2 composite structures
By 2A8Fab and cyno TFPI K1K2 with 1：1 mixed in molar ratio is to form compound.Use Superdex 200 columns carry out last purifying.Compound is condensed into 10.8mg/ml for structural research.Obtained in following two conditions The crystallization of compound containing 2A8Fab and TFPI K1K2：(1) 100mM HEPES pH7.5,12.5%PEG8000, it is produced Diffraction reachesAcicular crystal；(2) 100mM HEPES pH 7.5,1600mM ammonium sulfate, 2%PEG1000, its generation are spread out Penetrate and reachBulk crystals.Synchronization is remotely performed by the instantaneous low-temperature protection of crystal and in Advanced Photon Source Accelerator data is collected.Imaged frame is handled using software AutoPROC (Global PhasingLtd).The data belong to space group P3221, structure cell are as follows： α=β=90 °, γ=120 ° are each asymmetric single Position has a compound.Use the homology model of 2A8Fab and the structure for the TFPI K2 domains that can openly obtain (RSCBProtein Data Bank；PDBcodes1TFX and 4DTG) molecular replacement search is carried out to produce making us for each component The parsing that class is convinced.Refine is carried out using software PHENIX,The final R/Rfree factors be 0.1667 He respectively 0.2088, the RMSD of key areThe RMSD of angle is 1.474 °.Residue based on Fab TFPI K1K2 interfaces it is hidden The surface region (BSA) and BSA percentages (%BSA) of Tibetan, measure the epitope and paratope of the compound.Following TFPI's Residue in TFPI K1K2 domains participates in directly contacting 2A8Fab (according to the epitope of BSA)：D31、D32、G33、P34、C35、 K36, E100, E101, P103, G104, I105, C106, R107, G108, Y109, E123, K126, Y127 and G128.It is following Residue in the heavy chain of 2A8Fab includes heavy chain paratope：G26, T28, S31, Y32, Y96, R97, Y98, W99 and D101 (Kabat numberings).Residue in the light chain of following 2A8Fab includes Light Chain Antigen paratope：L28、R29、N30、Y31、Y32、 Y49, Y50, D51 and N66 (Kabat numberings).The epitope and BSA the and %BSA values of paratope residue are shown in table 21. Also the closely related antibody 2A8- in the compound with TFPI K1K2 is parsed using substantially identical method 200.The epitope and paratope of this antibody and 2A8's is consistent.
Table 21
Such as the hiding surface region (BSA) and BSA hundred of 2A8Fab/cyno TFPI K2 composite structures median surface residue Divide than anti-TFPI antibody 2A8 epitopes and paratope residue defined in (%BSA)
(antibody light chain (LC) and heavy chain (HC) residue use Kabat definition numbering) blocks (20Or the BSA of bigger, or Participate in electrostatic interaction) it is applied to BSA analyses
3.Mab 2974Fab/TFPI K2 composite structures
By (R＆D systems) Fab the and cyno TFPI of Mab 2974 K2 with 1：1.2 mixed in molar ratio is to form compound.Make Last purifying is carried out with 200 columns of Superdex.Compound is condensed into 17.5mg/ml for structural research.In 100mM lemons The crystal of the compound containing Mab 2974Fab and TFPI K2 is obtained in sour sodium pH5.6,20% isopropanol, 20%PEG4000, It produces diffraction and reachesBlock shape crystal.By the instantaneous low-temperature protection of crystal and long-range in Advanced Photon Source Perform synchrotron Data Collection.Imaged frame is handled using software AutoPROC (Global PhasingLtd).The compound Data belong to space group P212121, structure cell is as follows： α=β= γ=90 °, each asymmetry unit have three compounds.Since the sequence of Mab 2974Fab can not obtain, collect singleHigh-resolution data group, together with bioinformatic analysis to decode protein sequence.Use Mab 2974Fab Structure and can disclose obtain TFPI K2 domains structure (RSCBProtein Data Bank；PDBcodes1TFX and The parsing of compellent each component can be produced by 4DTG) carrying out molecular replacement search.Refine is carried out using software autoBUSTER, The final R/Rfree factors be 0.1702 and 0.2161 respectively, the RMSD of key isThe RMSD of angle is 1.13°.The hiding surface region (BSA) and BSA percentages (%BSA) of residue based on Fab TFPI K2 interfaces, measure The epitope of the compound.Residue in the TFPI K2 domains of following TFPI participates in directly contacting Mab 2974Fab (according to BSA Epitope)：E100、E101、P103、R107、Y109、T111、N116、Q118、S119、Q121、E123、R124、F125.Epitope BSA the and %BSA values of residue are shown in table 22.
Table 22
As Mab 2974Fab/cyno TFPI K2 composite structures median surface residue hiding surface region (BSA) and Anti- 2974 epitopes of TFPI antibody Mab block (20 defined in BSA percentages (%BSA)Or the BSA of bigger, or participate in electrostatic Interaction) it is applied to BSA analyses
Residue Chain Residue # Classification BSA %BSA
GLU TFPI K2 100 Epitope 25.9 16.8
GLU TFPI K2 101 Epitope 42.2 54.7
PRO TFPI K2 103 Epitope 26.8 58.5
ARG TFPI K2 107 Epitope 32.3 16.4
TYR TFPI K2 109 Epitope 83.1 62.9
THR TFPI K2 111 Epitope 13.8 18.9
ASN TFPI K2 116 Epitope 23.9 71.9
GLN TFPI K2 118 Epitope 107.0 62.7
SER TFPI K2 119 Epitope 48.4 87.6
GLN TFPI K2 121 Epitope 51.6 53.3
GLU TFPI K2 123 Epitope 61.6 79.8
ARG TFPI K2 124 Epitope 56.0 33.2
LYS TFPI K2 126 Epitope 114.8 91.8
4.TFPI-23Fab/cyno TFPI K2 composite structures
By TFPI-23Fab and cyno TFPI K2 with 1：2 mixed in molar ratio is to form compound.Use Superdex 200 columns carry out last purifying.Compound is condensed into 12.4mg/ml for structural research.In 100mM Bis-Tris The crystal of TFPI K2+4D8 Fab compounds is obtained in pH6.5,20%PEGMME5000.It produces diffraction and reachesFiber Shape crystallizes.Synchrotron data receipts are remotely performed by the instantaneous low-temperature protection of crystal and in Advanced Photon Source Collection.Imaged frame is handled using software AutoPROC (Global PhasingLtd).The data belong to space group P1, and structure cell is as follows：α=101.83 °, β=92.27 °, γ=96.78 °, it is each not Symmetrical units have the compound of six copies.Using TFPI-23Fab homology model and can disclose obtain TFPI K2 tie Structure (the RSCBProtein Data Bank in structure domain；PDBcodes1TFX and 4DTG) molecular replacement search is carried out to produce respectively The parsing for making us class conviction of component.Using software autoBUSTER progress refine, andThe final R/Rfree factors It is 0.1961 and 0.2344 respectively, the RMSD of key isThe RMSD of angle is 1.22 °.Based on Fab TFPI K2 interfaces BSA the and BSA percentages (%BSA) of the residue at place, measure the epitope and paratope of the compound.The K2 knots of following TFPI Residue in structure domain participates in directly contacting TFPI-23Fab (according to the epitope of BSA)：D102、I105、C106、R107、G108、 R112, Y127, G129, C130, L131, G132, M134 and E138.Under the residue that is listed in the heavy chain of 4D8Fab resist comprising heavy chain Former paratope：A33, W47, A50, I51, S52, S56, Y58, L95, G96, A97, T98, S99, L100 and S100A.It is following Residue in the light chain of 4D8Fab includes Light Chain Antigen paratope：A29, Y31, Y91, S95A, G95B and S95C.Epitope and antigen BSA the and %BSA values of paratope residue are shown in table 23.
Table 23
As TFPI-23Fab/cyno TFPI K2 composite structures median surface residue hiding surface region (BSA) and Anti- TFPI antibody TFPI-23 epitopes defined in BSA percentages (%BSA) and paratope residue (antibody light chain (LC) and Heavy chain (HC) residue uses Kabat definition numbering) blocks (20Or the BSA of bigger, or participate in electrostatic interaction) be applied to BSA analyzes
5.TFPI-24Fab/ macaque TFPI K2 are complicated
By TFPI-24Fab and cyno TFPI K2 with 1：2 mixed in molar ratio is to form compound.Use Superdex 200 columns carry out last purifying.Compound is condensed into 12.2mg/ml for structural research.In 20%PEG3350,200mM nitre The crystal of TFPI K2/TFPI-24Fab compounds is obtained in sour ammonium.It produces diffraction and reachesCrystal.Crystal is instantaneous Low-temperature protection simultaneously remotely performs synchrotron Data Collection in Advanced Photon Source.Use software AutoPROC Handle imaged frame.The data belong to space group P212121, and structure cell is as follows： In,α=β=γ=90 °, each asymmetry unit have a compound.Use the homologous of TFPI-24Fab Model and structure (the RSCBProtein Data Bank that the TFPI K2 domains obtained can be disclosed；PDBcodes1TFX and Molecular replacement search 4DTG) is carried out to produce the parsing of compellent each component.Refine is carried out using software autoBUSTER, The final R/Rfree factors be 0.1900 and 0.2269 respectively, the RMSD of key isThe RMSD of angle is 1.18°.The hiding surface region (BSA) and BSA percentages (%BSA) of residue based on Fab/TFPI K2 interfaces, measure The epitope and paratope of the compound.Residue in the K2 domains of following TFPI participates in directly contacting TFPI-24Fab (according to the epitope of BSA)：E100、E101、D102、G104、I105、C106、R107、G108、Y109、I110、G129、C130、 L131 and G132.Residue in the heavy chain of following TFPI-24Fab includes heavy chain paratope：A33、Q35、W47、G50、 I51, S52, N53, R55, S56, I57, G58, F95, L96, H97, S99 and D101.It is residual in the light chain of following TFPI-24Fab Base includes Light Chain Antigen paratope：M31, Y32, H34, Y36, L46, R50, W91 and Y96.Epitope and paratope residue BSA and %BSA values are shown in table 24.
Table 24
As TFPI-24Fab/cyno TFPI K2 composite structures median surface residue hiding surface region (BSA) and Anti- TFPI antibody TFPI-24 epitopes defined in BSA percentages (%BSA) and paratope residue (antibody light chain (LC) and Heavy chain (HC) residue using Kabat definition numbering) block (Or the BSA of bigger, or participate in electrostatic interaction) be applied to BSA analyzes
The epitope analysis of 6.hz4F36
The structure of hz4F36fab in compound with mankind's TFPI K2 domains can be from Protein Data Bank Obtain (PDB registration number 4DTG).BSA and BSA percentages based on the interface residue in hz4F36/TFPI K2 composite structures (%BSA), defined epitope residues are shown in table 25.
Table 25
Such as the hiding table of hz4F36Fab/cyno TFPI K2 composite structures (PDB registration number 4DTG) median surface residue Anti- TFPI antibody hz4F36 epitope residues defined in face region (BSA) and BSA percentages (%BSA).Block (Or bigger BSA, or participate in electrostatic interaction) be applied to BSA analysis
7. the comparison of anti-TFPI antibody epitopes
The epitope of anti-TFPI antibody shown in comparison sheet 20 to 25 in table 26 and table 27.The display of table 26 is specific to TFPI The epitope of the antibody of K2 domains.Table 27 includes the antibody (2A8 and 2A8-200) other 2 in combination with K1 and K2 domains.
Table 26
Anti- TFPI antibody epitopes residue based on data in table 20,22-25
X represents the TFPI amino acid residues of a part for epitope.X (runic) represents the Novel clock of antibody disclosed by the invention Position residue.Note that TFPI-23 will not with hz4F36,4D8 or mab2974 competition binding TFPI, but really with TFPI-24 compete With reference to TFPI.Antibody 2A8 and 2A8-200 do not include in this table, because they need both K1＆K2 domains for combining And K2 domains are not combined individually.
Table 27
The more anti-TFPI antibody epitopes residue of data based on table 20-25
X represents the TFPI amino acid residues of a part for epitope.X (runic) represents the Novel clock of antibody disclosed by the invention Position residue.Antibody 2A8 and 2A8-200 need both K1＆K2 domains and are tied for combining and not combining individually K2 or K1 at the same time Structure domain.
Table 28
Key is predicted using the method (Accelrys Discovery Studio 4.1) calculated by Alanine-scanning TFPI epitope residues are used for TFPI-23
According to>Any threshold of 1kcal/mol, prediction are mutated when 3 TFPI residues (Ile105, Arg107 and Leu131) For alanine when combination to TFPI-23 and TFPI there is notable contribution.
Table 29A
In TFPI K2 epitopesIn the range of TFPI-23CDR and Framework residues
Using Forecasting Methodology (Accelrys Discovery Studio 4.1) is calculated, display that be predicted will not be negative Influence the stability of TFPI-23 or each CDR/ paratopes residue to the compatibility of TFPI possible 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (< 0.5kcal/mol, the 4th to 6 row).These are divided into three groups：(1) to combine with least neutral influence (<0.5kcal/ Mol compatibilities, the 4th row), (2) to combine with neutral/influence for stablizing (<- 0.5kcal/mol compatibilities, the 5th row) and (3) There is preceding 3 predictions site of most stable of influence on compatibility.All residues are using Kabat numberings (the 6th row). 138Glu/L93Ser and 131Leu/L96Gly due to distance only slight beyond(it is respectivelyWith), but it is close enough(it is after rounding up), it is included as selectable contact residues.
Table 29B
TFPI epitope residues and corresponding TFPI-23 paratopes residue
Table 29C
Pass through the TFPI epitope residues and corresponding TFPI-23 paratopes residue of BSA (without minimum BSA applications)
Embodiment 11. dilutes prothrombin time (DPT)
The blood plasma that anti-TFPI antibody suppresses mankind FVIII and lacks is studied using prothrombin time (PT) measure is diluted The ability of endogenous TFPI in (hemophilia A).Dilution PT is to extend the clotting time using diluted tissue factor (Innovin) Modification PT detection.
, will in terms of dPT analyses in the blood plasma (George KingBiomedical) that mankind FVIII lacksReagent is in dilution buffer agent (imidazoles 50mM, sodium chloride 0.1M, BSA 1mg/ml, CaCl28.34mM, pH 7.4) In with 1：3000 dilution and preincubate to 37 DEG C.Before the assay soon, blood plasma is thawed 5 minutes in 37 DEG C of water-baths.In PBS Prepare the dilution of anti-TFPI antibody and add in blood plasma, then by the blood plasma in incubation at room temperature 20 minutes.After incubation, by 50 μ L's Blood plasma 37 DEG C be incubated 1 minute, add 50 μ L be warmed to 37 DEG C with 1：3000 is dilutedOriginated immediately after reagent Coagulation test.In 37 DEG C of usesBlood coagulation analyzer carries out the time for reaching blood coagulation.Data point is collected, is repeated twice, Enter data into Microsoft Excel and use GraphPadEstimate 50% valid density (EC50).The results show In table 30.
TFPI lowers the exogenous FVIIa/TF/FXa approach of blood coagulation, reduces generation FXa and finally reduces fibrin ferment.dPT Measure the influence of exogenous coagulation pathway.Data are shown adds anti-TFPI antibody meeting dose-dependant in hemophilia A plasma Shorten the clotting time in ground.The control IgG of 300nM does not influence the clotting time.
Table 30
12 thrombelastogram of embodiment (THROMBOELASTOGRAPHY) (TEG)
Thrombelastogram (TEG) is to measure dynamic (dynamical) hemostasis measure comprehensively that clot in whole blood is formed.From healthy human Donor isolates whole blood and is pumped into the plastic blood collecting pipe containing 3.2% sodium citrate and makes the Activated Coagulation agent example of introducing Such as the blood that tissue factor is minimum, and the first pipe of discarding is extracted out.With control mice anti-human IgG2 (100 μ g/ml) or with inhibition FVIII antibody (GM1805 (Green Mountain), 100 μ g/ml) handle the Citrated whole blood 1 it is small when to suppress endogenous Property FVIII, induce hemophilia A sample phenotype.The whole blood (320 μ L) that the dosage of anti-TFPI antibody or IgG1 control antibodies will be given adds EnterIn reaction cup, 0.2M calcium chloride and 20 μ L containing 20 μ L in the reaction cup in 20mM HEPES, 150mM chlorinations Diluted esterified tissue factor in sodium, pH 7.4And cause in each reaction finally with 1:200,000 is dilute The esterified tissue factor dilution released.Reaction is immediately begun to reruning twice when whole blood is added TEG glasss.According to The instruction of manufacturer utilizes after being calibrated with I level and the control of Section II levelSoftware carries out on 5000 hemostasis analyzers of TEG Analyze (Haemonetics).The reaction 60 minutes is carried out at 37 DEG C.Referring to table 31.
Table 31
TEG parameters in hemophilia A (HA) blood of the antibody induction handled with anti-TFPI antibody
Table 31 is shown significantly extends TEG-R values up to 41.5 minutes with FVIII antibody processing whole blood.In the presence of whole blood, Anti- TFPI 106 shows the favourable spectrum relative to 2A8-200 and 4F36.Add TFPI-106,2A8-200 or 4F36 (300nM) causes TEG-R values respectively up to 17.85 minutes, 20.4 minutes and 24.05 minutes.By TEG-R values decline and observe The increases of TEG- α angles show that anti-TFPI 106 promotes hemophilia blood clotting.
Generated in embodiment 13. with TFPI and fibrin ferment
Utilize two kinds of chromogenic assay, direct factor xa activity measure and two based on TFPI suppression TF-FVIIa generations FXa Stage FVIIa/TF/FXa measure is measured by TFPI antibody and TFPI.The first measure in, by various concentrations (0 to TFPI-106, TFPI-118, hz4D8 and the mankind of two kinds of reference antibody 4F36 or 2A8-200 and fixed concentration weight 500nM) Group TFPI K1K2 and FXa preincubates to form compound to allow.Use colour developing FXa substrates assessment FXa activity.In this measure The TFPI antibody of the addition present invention causes the increase of FXa active doses dependence, and (referring to table 32, Xa suppresses measure, EC50Value).
In vivo, the TFPI of two kinds of principal modes is TFPI- α (K1K2K3) and TFPI- β (K1K2).In two benches The ability that TFPI antibody suppresses restructuring TFPI K1K2 or TFPI K1K2K3 is assessed in FVIIa/TF/FXa measure.The measure measures Neutralize the merging effect that TFPI suppresses FXa and FVIIa/TF/FXa.By the antibody of progressive concentration (0 to 500nM) together with TFPI It is incubated, adds using FXa chromogenic substrates to measure in the measure of FVIIa/TF/FXa and FXa activity.The TFPI antibody of the present invention Suppression that TFPI K1K2 activate the FX that is mediated by FVIIa/TF is neutralized (referring to, table 32, FVIIa/TF/Xa EC50Value).This hair Bright exemplary antibodies also can effectively suppress TFPI K1K2K3 (TFPI-106 be used for neutralize TFPI K1K2K3 to FVIIa/ The EC of the suppression of TF/FXa50It is 8.47nM).The data prove total length and the suppression of truncated TFPI.
Haemophiliachemophiliac clinical severity is related with the residual level of coagulation factor activity.<1% factor active and heavy table Type is related, and osculant hemophilia is related to 2 to 5% factor active, and it is light-duty and>5% to<40% factor active is related). The defects of haemophiliachemophiliac intrinsic coagulation pathway, causes fibrin ferment generation deficiency.Experiment (TGA) is generated using fibrin ferment to check To the inhibitory action of the endogenous TFPI in the haemophilic plasmas of platelet poor.TGA measure measurement fibrin ferments generate first Stage beginning, activation stage and deactivation phases.Utilize the automatic fibrin ferment (Calibrated Automated Thrombin) of calibration (CAT) generation measure test TFPI antibody recovers the ability of the fibrin ferment generation in the haemophilic plasmas of platelet poor.Will TFPI-106, TFPI-118, hz4D8 and two kinds of reference antibody 4F36 or 2A8-200 (0 to 500nM) are incubated in haemophilic plasmas Educate to neutralize TFPI, add in the measure.Compared with the mixing of normal human subject (pooled) blood plasma, human hemophilia's blood plasma In fibrin ferment generation substantially reduce.When by normal control FACT (it is 1U/mL FVIII that its is normalized) mix hemophilia A blood Dose dependent response can be observed when in slurry.Similarly, the B structure domain deletion form FVIII for adding 200ng/mL (1U/mL) can Recover fibrin ferment generation and reach 100nM.In the measure time-histories of 60 minutes, minimum fibrin ferment life is observed in haemophilic plasmas Into.Blood plasma is incubated together with the antibody of the present invention causes peak value fibrin ferment, intrinsic coagulation enzyme potential and Rate Index dosage Rely on increase.Also reference antibody 2A8-200 and 4F36 is measured for comparing (table 32, TGA speed EC50Value).
Internal effect of the embodiment 14. in hemophilia mouse model
Some anti-TFPI antibody are tested as the effect of Procoagulants by the use of the cross-section measure of acute afterbody in hemophilia mouse Power.In the measure, the distal portions of tail, which are blocked, causes massive blood loss, if applying hemostasis soon before cutting or after cut-out Agent can be reduced and lost blood.Hemophilia mouse receives the anti-TFPI antibody (6mg/kg) that volume is 4ml/kg, non-specificity through tail vein IgG compares single dose intravenous (IV) dosage of (6mg/kg) or saline vehicle.The different time upon administration, according to following assessment antibody Influence to bleeding.
By intraperitoneal routes with ketamine/xylazine mixture by mouse anesthesia.By 37 DEG C of tail immersion, 50 2 minutes in the phosphate buffered saline (PBS) (PBS) that milliliter warms in advance.The afterbody for manufacturing 3mm is cross-section and collect blood into PBS totally 10 Minute.Using following technology by measuring the content of hemoglobin of the PBS come volume of quantitatively losing blood.Test tube is centrifuged red to collect Cell, is resuspended in 5 milliliters of cracking buffer (8.3g/l ammonium chlorides, 1.0g/l saleratus and 0.037g/l EDTA) simultaneously By spectrophotometer measurement sample 575nM absorbance.Absorption values are changed into (the μ that always loses blood using standard curve L).By variance analysis (ANOVA), reuse GraphPad Prism softwares and carry out Dunnett multiple comparative tests to assess The statistical significance of difference between various modes.As a result represented with the standard deviation (SEM) of average value ± average value.Below In the attached drawing of description, statistical significance is defined as P values<0.05, and indicated by asterisk above data.
Fig. 3 A show that compared with supporting agent compares (brine), the different time before afterbody is cross-section gives 2A8-200 antibody agents Measure the influence lost blood after afterbody is cross-section to hemophilia A mouse (be expressed as FVIII-/-).Fig. 3 B, which are shown, compares (salt with supporting agent Water) and non-specific human IgG 1 (be expressed as hIgG1) compare, the different time before afterbody is cross-section gives 2A8 antibody dosages To the influence lost blood of the hemophilia A mouse (be expressed as FVIII-/-) after afterbody is cross-section.Fig. 3 B be also shown in afterbody it is cross-section before Two different times give the shadow lost blood of the 2A8 antibody dosages to normal mouse (be expressed as FVIII+ /+) after afterbody is cross-section Ring.Fig. 3 C show that compared with supporting agent compares (brine), the different time before afterbody is cross-section gives antibody 4D8,21,23,24 dose Measure the influence lost blood to hemophilia A mouse after afterbody is cross-section.Fig. 3 D are shown compared with supporting agent compares (brine), horizontal in afterbody Different time before disconnected gives the influence lost blood of 106 dosage of antibody to hemophilia A mouse after afterbody is cross-section.Fig. 3 E show with Supporting agent control (brine) is compared, and it is horizontal in afterbody to hemophilia A mouse that the different time before afterbody is cross-section gives 118 dosage of antibody The influence lost blood having no progeny.Fig. 4 shows that compared with supporting agent compares (brine), the different time before afterbody is cross-section gives antibody The influence lost blood of 106 dosage to factor Ⅸ deficient mouse after afterbody is cross-section.
As shown in Fig. 3 D and Fig. 4, in acute traumatic injury model, when antibody is the difference before afterbody transection lesion When time applies, the hemophilia A or factor Ⅸ deficient mouse of the anti-TFPI antibody 106 through applying 6mg/kg are lost blood reduction.In hemophilia In A mouse, the haemostatic effect may persist to after application the youthful and the elderly up to 189 it is small when, although after application 240 it is small when the effect recover Into control level.In factor Ⅸ deficient mouse, the effect may persist to after application the youthful and the elderly up to 72 it is small when and it is 192 small after application When revert to baseline values.
Above-mentioned data and the result shows that the antibody (including antibody TFPI 106) of the test be applied to preventability blood friend The object of sick A or hemophilia B before traumatic damage or other kinds of bleeding episode to reduce bleeding.
Also test antibody TFPI 106 in hemophilia A mouse as the effect of hemostat to measure when cross-section in afterbody after Whether apply soon can reduce bleeding.This is carried out using method of the test in the effect of the cross-section preceding administration of antibodies of afterbody is similarly used for A little experiments, but immediately by being inserted into jugular catheter infusion TFPI 106 (6mg/kg) or recombinant factor after afterbody is cross-section The IV dosage of VIII (200 units/kg), then, collects blood 10 minutes quantitatively to lose blood again.In second group of experiment, pressing from both sides Firmly then, received respectively using the TFPI 106 and Factor IX (200U/kg) 2 minutes of hemophilia A mouse various dose after tail Collection blood is quantitatively lost blood for 10 minutes again.
Fig. 9 A show that compared with supporting agent compares, the antibody TFPI 106 after afterbody is cross-section immediately using 6mg/ml can be effective Losing blood in hemophilia A mouse is reduced, although what the Factor IX for being not reaching to the 200U/kg applied in the same fashion was obtained Same degree.Fig. 9 B are reduced with showing after the cross-section damage of afterbody 106 2 minutes dose responses of administration of antibodies TFPI The bleeding of hemophilia A mouse, and when antibody TFPI 106 and recombinant factor VIII 2 minutes are administered simultaneously after cross-section in afterbody, It is about same effective with the recombinant factor VIII of 200U/kg in the TFPI 106 for the maximum dose level (6mg/kg) tested.These figures Described in data and the result shows that antibody TFPI 106 can be effective as opening because of wound or some other reasons The demand property treatment (on-demand treatment) of the object of beginning bleeding.
15. pharmacokinetics of embodiment and product catabolism
By vein (IV) and/or subcutaneous (SC) approach give after dosage Wistar Han rats, New Zealand White Rabbit and The pharmacokinetics (PK) and/or Toxicokinetics (TK) of TFPI-106 is characterized in machin.
The quantitative anti-TFPI in the rabbit blood plasma of citric acid sodium of sandwich immune measure is used on Gyrolab.It is logical Gyrolab instruments are crossed, reacton is read in 1% photomultiplier (PMT) device.By interpolation method from standard curve determination sample This concentration, the standard curve are fitted using 5 parameter logistic curves for mixing 1/y2 reaction weightings.Contain 5% citric acid collected Standard point range in the measure buffer of sodium rabbit blood plasma is 0.78ng/ml to 891ng/ml, and in 100% rabbit blood plasma Quantification range in matrix is 90ng/ml to 5500ng/ml.Using containing the 5% citric acid sodium rabbit blood plasma collected Five anti-TFPI samples (concentration 0.45,8.0,47.15,153,275ng/ml) in measure buffer are used as quantitative control.
It is also quantitative anti-in citric acid sodium rat plasma using sandwich immune measure on Gyrolab according to above-mentioned TFPI.Containing the 5% citric acid sodium rat plasma collected measure buffer in standard point range for 0.78ng/ml extremely 891ng/ml, and the quantification range in 100% rat plasma matrix is 90ng/ml to 5500ng/ml.Using containing 5% Five in the measure buffer of the citric acid sodium rat plasma collected anti-TFPI samples (concentration is 0.45,8.1,47.15, 153rd, 275ng/ml) it is used as quantitative control.
Measured using the quantitative ligand bindings of whole mankind Ig using Meso Scale Discovery (MSD) measure platforms The quantitative anti-TFPI antibody in machin blood plasma.With the anti-Human IgG Fc antibody's inspection of (ruthenylated) mouse of ruthenium The anti-TFPI antibody combined is surveyed with generation electrochemical luminescence (electrochemiluminescence) signal in MSD instruments. By interpolation method from the standard curve determination concentration of specimens being fitted using 5- parameter logistics equation, the weighting for standard curve is public Formula is 1/y^2.The anti-TFPI antibody that standard point range in 5% monkey plasma is 0.999ng/ml to 1156ng/ml, and Quantification range in 100% blood plasma is 64.8ng/ml to 7136ng/ml.By five in 100% blood plasma, concentration 64.8, 117th, the anti-TFPI antibody samples of 680,3964 and 7136ng/ml are diluted to 1:20 MRD (be 3.24 respectively in 5% blood plasma, 5.83rd, 34.0,198 and 357ng/ml) it is used as quantitative control.
Compared with isotype controls monoclonal antibody (mAb), TFPI-106 shows faster clear in New Zealand White Rabbit Except rate (CL).In machin, TFPI-106 shows non-linear PK dynamics in low dosage, and observed by anti-TFPI antibody The drug distribution (TMDD) for the target mediation arrived is consistent.Referring to table 32.
Table 32 summarizes the exemplary anti-TFPI antibody (hum4D8, TFPI-106 and TFPI-108) of the present invention and two refer to The some pharmacokinetic properties and pharmacological activity of antibody (4F36 and 2A8-200).
Table 32
The pharmacokinetic property and pharmacological activity of exemplary anti-TFPI antibody
16. antibody of embodiment suppresses the anastalsis reinforcement of TFPI in the hemophilia mouse damage model of induced with laser Interior platelet accumulation and fibrin generation
Such as the statement of elsewhere of the present invention, tissue factor approach restrainer (TFPI) be directly in conjunction with and suppress tissue factor (TF)/factor VIIa/Factor Xa complex simultaneously regulates and controls the plasma serine protease inhibitor originated by the blood coagulation of TF inductions.Resistance Disconnected TFPI can potentially promote the hemostasis started by TF/FVIIa, compensate in hemophilia A or B Factor IX (FVIII) or because The loss of the sub- IX factors.The antibody of the present invention suppresses TFPI with extensive cross-species reactivity.Herein, using intravital microscope (IVM) hemostasis effects of the TFPI-106 to internal platelet thrombosis and fibrin deposition is assessed in hemophilia A and B mouse Fruit.
Materials and methods：TFPI-106 antibody, recombinant factor VIII and supporting agent are prepared according to the description of previous elsewhere of the present invention (brine of phosphate-buffered).The anti-CD42c antibody of Dylight-649 is obtained from Emfret Analytica (Germany).According to The specification (Life Technologies, Carlsbad, CA) of manufacturer uses Protein Labeling Kit, with Alexa 488 labeled fibers protein antibodies of Fluor clone 59D8 (Hui KY et al. (1983) Science 222 (4628):1129- 1132).Obtaining average weight from the proprietary line for being maintained at Charles Riverlaboratories (Wilmington, MA) is 30 to 35 grams of male hemophilia A mouse (F8KO).Mouse is set at least to adapt to environment 3 days before experimental arrangement.
Give male hemophilia A, hemophilia B or C57BL/6J wild types (WT) mouse TFPI-106 (6mg/kg), supporting agent, TFPI-106 (0.7mg/kg) single dose intravenous agent of recombinant human FVIII (rFVIII, 200IU/kg) or Alexa-488 marks Amount.Use the cremaster microcirculation of the mouse of IVM observation anesthesia.After laser thermal damage is carried out to the vascular wall of cremasteric artery Quantitative platelet accumulation and fibrin generation.Blood platelet is visualized and led to using the anti-CD42c of Dylight-649 (GP1b β) Cross Alexa-488 antifibrins clone's 59D8 detection fibrins.
More specifically, in order to prepare the hemophilia A mouse for intravital microscope imaging, pass through intraperitoneal delivery chloramines Ketone/xylazine mixture is by Animal Anesthesia.It is intubated in jugular vein and uses yellow Jackets (5mg/kg, intravenous) to maintain Anesthesia.In trachea cannula to keep airway patency.Then, cremaster exposure is made to make capilary visualize.In whole imaging phase Between animal is maintained on warm heating cushion, and with the cremaster tissue of warm buffer immersion exposure.Led by neck Pipe infusion needle is to 649 CD42c of antibody Dylight (GP1b β) of hematoblastic mark and will not intersect instead with fibrinogen The fibrin antibody (488 anti-fibrin clone 59D8 of Alexa Fluor) answered.Use the laser beam of focusing (532nM) starts damage on mouse cremaster capilary.Every mouse receives 2 damages by induced with laser.Without place First damage is manufactured in the mouse of reason as control.Second is manufactured in same mouse to damage and pass through neck conduit immediately Intravenous administration TFPI-106 (6mg/kg).Monitored from the fluorescence intensity of platelet accumulation and fibrin deposition in two damages Clot is formed.
Collect data point (fluorescence intensity) and analyzed using SlideBook softwares (version 6.0).It is strong to draw median fluorescence Spend the function of time as each blood coagulation and useSoftware (start context 6.03) measurement is corresponding Area under the curve.UseSoftware (start context 6.03), measure system is examined using Mann-Whitney The conspicuousness of meter.
As a result：Blood in the TFPI-106 detection WT mouse marked using Alexa 488 in thrombocytic blood clot and endothelium is small The TFPI (Fig. 5) of plate aggregate site.Fig. 5 A include the figure (numbering 1 to 6) six on the top, it, which shows, is applying control IgG In the WT mouse of (being marked using Alexa 488), using 0 second after Laser Induced Damage (Fig. 5 A-1), 15 seconds (Fig. 5 A-2), 30 seconds Blood is detected with 649 CD42c of Dylight after (Fig. 5 A-3), 60 seconds (Fig. 5 A-4), 90 seconds (Fig. 5 A-5) and 120 seconds (Fig. 5 A-6) Platelet.Do not detect and (detected using 649 CD42c of Dylight) in the platelet thrombus of damage location and marked to Alexa 488 Note.Fig. 5 B are included in six figures (numbering 1 to 6) of the figure bottom, it shows uses Laser Induced Damage in WT mouse About 15 seconds afterwards, it can start to detect the TFPI-106 (Fig. 5 B-2) that Alexa 488 is marked in the platelet thrombus along endothelium, Fluorescence intensity gradually increases from about 30 seconds (Fig. 5 B-3), 60 seconds (Fig. 5 B-4) and 90 seconds (Fig. 5 B-5), and then continuing force is to about 120 seconds (Fig. 5 B-6), the wherein blood platelet are detected using the CD42c marked of Dylight 649.
About 0.5 it is small when with supporting agent processing hemophilia A mouse compared with, TFPI-106 strengthen in hemophilia A (F8KO) mouse In platelet accumulation and fibrin generation and the lasts about 168 it is small when.More specifically, in TFPI-106 processing In F8KO mouse (hemophilia A), IVM shows damage location green fluorescence (488 antifibrins of Alexa when about 0.5 is small Clone 59D8) and red fluorescence (the anti-GP1b β detections blood platelets of CD42c that Dylight 649 is marked) increase, it lasts about 168 Hour and the fluorescence mode be similar to supporting agent processing WT mouse what is observed in.However, in the hemophilia A of supporting agent processing In mouse (wherein both do not observed platelet accumulation or do not observed fibrin generation) and it is not detected by this kind of fluorescence.
Hemophilia A mouse shows platelet accumulation (Fig. 6 A) and fiber when after giving TFPI-106 dosage about 0.5 is small Proteinosis (Fig. 6 B) increase, similar to the level (*=P reached by rFVIII<0.005 instruction is on each figure). TFPI-106 processing hemophilia A mouse haemostatic effect last up to 168 it is small when.Similarly, the hemophilia B of TFPI-106 processing Mouse also showed that compared with supporting agent compares, the hemostasis improved in about 30 minutes upon administration, and platelet accumulation and fiber egg White generation increase.Receive TFPI-106 and rFVIII dosage hemophilia mouse none reach and seen in non-hemophilia WT mouse The highest level of the fibrin deposition observed.Therefore, for the as shown by data after laser hazard endothelium, TFPI can be in damage location Detect.Importantly, the data prove that being administered to hemophilia mouse TFPI-106 can cause to show in damage from laser model Write and lasting hemostasis improves.
17. anti-TFPI of embodiment suppresses influence of the combination in hemophilia A and B TGA to fibrin ferment generation with FVIIA
Method：Fibrin ferment generation experiment (TGA)
The single anti-TFPI antibody TFPI-106 of experiment (TGA) assessment is generated using fibrin ferment and its with recombinating FVIIa (rFVIIa) influence of the combination in haemophilic plasmas to fibrin ferment generation.From from George King Biomedical The donor of the inadequate natural endowment of (Overland Park, KS) and HRF (Raleigh, NC) obtains the platelet poor of Citrated Heavy hemophilia A (FVIII shortages), heavy hemophilia A or hemophilia B (FIX shortages) blood plasma with inhibitor.From George King Biomedical obtain normal pooled plasma (non-hemophilia).Fibrin ferment generates reagent；PPP- reagents LOW is (final It is 4 μ l phosphatide and 1pM tissue factors in reaction)；FluCa buffers (containing calcium chloride and fluorogenic substrate) and from The thrombin calibrator that Diagnostica Stago (Parsippany, NJ) are obtained.Using including Fluoroskan Ascent The automatic fibrin ferment life of the calibration of fluorescence disc type analyzer (Thrombinoscope BV, Maastricht, Netherlands) Fibrin ferment generation experiment is carried out into figure (CAT).
By concentration, up to the restructuring FVIIa of 20 μ g/mL, (rFVIIa is pure in 20mM HEPES, 150mM sodium chloride, 1% ox blood Albumen (BSA), pH7.4) add in the hemophilia A plasma of 70 μ l.By the PPP- reagents LOW of 20 μ l and the anti-TFPI 106 of 10 μ l Add in reacting hole, it is 16 μ g/mL to make ultimate density.The calibration of reference control reaction include 20 μ l thrombin calibrator and 70 μ l blood plasma.Carry out 0 μ g/mL of test concentrations using supporting agent (brine (PBS) of phosphate-buffered).10 μ l supporting agents are added and refer to school In the hole of quasi- agent.In second group of reaction, the rFVIIa (5 μ l) in HEPES buffers or HEPES buffers (5 μ l) are added In the haemophilic plasmas sample (hemophilia A, the hemophilia A or hemophilia B with inhibitor) for entering 70 μ l, make in blood plasma The ultimate density of rFVIIa is 2 μ g/ml.By the anti-TFPI 106 of 20 μ l PPP reagents LOW and 5 μ L (in PBS dilute) or PBS (5 μ l) adds the haemophilic plasmas sample (75 μ l) through giving rFVIIa dosage and the haemophilic plasmas of HEPES buffers processing In (75 μ l), it is 16 μ g/mL to make ultimate density.In addition, measure is in the non-haemophilic plasmas (75 μ l) of HEPES buffers processing Anti- TFPI 106 (16 μ g/mL).The analysis includes undressed non-haemophilic plasmas (80 μ l) control.Run parallel ginseng Examine calibration reactions (hemophilia of 20 μ l thrombin calibrators and 80 μ l supporting agent dosage or non-haemophilic plasmas, or 80 μ l are without place The non-haemophilic plasmas of reason).It is all to react to rerun twice.Sample is incubated 5 minutes at 37 DEG C, then passes through addition The FluCa of 20 μ l makes total reaction volume be that 120 μ l react to start.It is every at 37 DEG C on Fluoroskan Ascent fluorescence photometers The fluorescence of blood plasma reaction was read every 20 seconds and compared with reference to thrombin calibrator reaction, to measure concentration of thrombin.37 DEG C, the intensity of fluorescence signal is persistently monitored using CAT.
As a result：Clotting factor FVIII (hemophilia A) and FIX (hemophilia B) lack hinder generate enough fibrin ferments by Fibrinogen is converted into fibrin with the clot of development stability.Anti- TFPI-106 (one kind specifically bind and suppress and/or Neutralize the novel monoclonal antibody of the inhibitory activity of TFPI) the exogenous tissue factor/FVIIa coagulation pathways of targeting.Receive TFPI- 106 patient with inhibitor is also subjected to rFVIIa to treat break-through bleeding.Therefore, in the presence with or without rFVIIa Under, the TGA (fibrin ferment generation experiment) approved using this area checks TFPI-106 to solidifying in haemophilic plasmas in vitro The influence of hemase generation.
Citrated platelet poor Factor IX lack human plasma in using fibrin ferment generation test into Row in vitro study with study in the presence of rFVIIa TFPI-106 activity.In a research, by fixed concentration (16 μ g/mL) The anti-TFPI 106 of (rFVIIa of this known concentration can increase the fibrin ferment generation in hemophilia A plasma) adds Factor IX and lacks In weary human plasma.The rFVIIa of the progressive concentration of most 20 μ g/mL is added in measure.Surprisingly, TFPI-106 Combination with rFVIIa recovers fibrin ferment and generates to horizontal what is observed in normal plasma.With TFPI-106 and scope 0.2, The peak value level of thrombin that the combination of the rFVIIa (eptacog alfa, eptacog alfa) of 2 and 20 μ g/mL is observed is similar. Data as shown in Figure 7 prove that TFPI-106 can improve the fibrin ferment of the heavy hemophilia A plasma through giving rFVIIa dosage Reaction of formation (Fig. 7, filled black, Dark grey and light grey line).
Also measurement TFPI-106 (the 16 μ g/mL in non-haemophilic plasmas (it is by with the clotting factor fully supplemented)；Figure 7, Dark grey dotted line) influence to fibrin ferment generation.Consistent with the TFPI extrinsic pathway inhibitory activity of TFPI-106, addition is anti- TFPI 106 causes fibrin ferment generation increase, is reduced with time lag and peak value fibrin ferment increases.Add TFPI-106 and rFVIIa Peak value fibrin ferment It is in the reporting range of normal non-haemophilic plasmas.
In other hemophilia A plasma (Fig. 8 A；1pM tissue factors and 4 μM of phosphatide), hemophilia B plasmas (Fig. 8 C；3BU presses down Preparation) and with inhibitor hemophilia A plasma (Fig. 8 B；3BU inhibitor) in further research in depositing with and without rFVIIa Under, the TFPI inhibitory activity of TFPI-106 graphically illustrates in Fig. 8 A, Fig. 8 C and Fig. 8 B the result that fibrin ferment generates respectively In.TFPI-106, which is added in the blood plasma, makes ultimate density be 16 μ g/mL.In these researchs, the ultimate density of rFVIIa is 2 μg/ml.Compared with supporting agent compares, 2 μ g/mL rFVIIa are added in haemophilic plasmas causes fibrin ferment generation appropriateness increase.With Add single rFVIIa to compare, add the group of single 16 μ g/mLTFPI-106 or 16 μ g/mL TFPI-106 and rFVIIa Conjunction can cause fibrin ferment generation increase, including higher peak value concentration of thrombin and the time lag of shortening.With rFVIIa and TFPI- It was observed that the minimum addition effect of fibrin ferment generation after 106 are jointly processed by.With single 16 μ g/mL TFPI-106 or 16 μ g/mL Peak value level of thrombin that the combination of TFPI-106 and rFVIIa is obtained with those of observed in non-haemophilic plasmas Quite, and no more than in level what is observed in the non-haemophilic plasmas of TFPI-106 dosage of giving.These data prove TFPI-106 can effectively be bypassed in hemophilia A, hemophilia B and be there is the fibrin ferment in the hemophilia A plasma of inhibitor to generate Lack.
Procoagulant activity in 18. human hemophilia's blood of embodiment and blood plasma
The present embodiment description using the whole blood and blood plasma that derive from the human subjects with hemophilia A and B when being tested, antibody The ratio of TFPI-106 and recombinant blood coagulation factor VIII and the styptic activity of factors IX.
Material and method.Under the scheme of institutional review board approval, derived from over the aspiration haemophiliac of 18 years old Whole blood and blood plasma (rich in blood platelet and platelet poor).The object have osculant or heavy Factor IX (FVIII) or Factor IX/factor (FIX) lack, with or without inhibiting antibody, but be in other respects health and be non-bleeding state. If volunteers are interior once using any factor replacement therapy, with active hemorrhage or with thrombus when 48 is small before entering research The medical history or family history of formation then foreclose them.In 11 volunteers, 5 are that heavy FVIII lacks, and 2 are Osculant FVIII lacks, and 1 is that osculant FIX lacks and 3 are that the heavy FVIII with FVIII inhibitor lacks.In order to Complete all targets of the research, by sterile venipuncture from the blood (10 pipe blood) of each volunteer 46mL enter containing In the vacuum tube of 3.2% sodium citrate.
Tester includes TFPI 106, the matched anti-human IgG of negative control group isotype1, recombinant human Factor IX And recombinant human factors IX, according to experiment, it is added into whole blood or blood plasma.According to the measure, test 1,5,20,50 or The TFPI 106 of 100nM, the control IgG of 100nM1Antibody and 5%, 10% or 40% restructuring that can reach normal Factor activity FVIII or FIX horizontal (being measured based on activated partial factor I (thromboplastin) time (aPTT)).In order to Concentration needed for acquisition, with dilution buffer agent, pH 7.4 (includes 20mM HEPES, 150mM NaCl and 0.5% bovine serum albumin Dilution test product in vain).
Procoagulants (procoagulant) effect of the trial target, including rotation are measured using the measure of three types Thrombus elasticity measure (ROTEM), fibrin ferment generation experiment (TGA) and dilution prothrombin time (dPT) measure.
ROTEM measure the whole blood sample under low shear conditions blood coagulation when viscoelastic properties.When blood coagulation carries out, the sample This viscosity increase, this can pass through pattern analysis.Using ROTEM analyzers (Pentapharm GmbH, Munich, Germany), ROTEM is carried out using Pentapharm softwares 1.0.04 to assess the blood coagulation in whole blood.By 0.300mL's 0.020mL is added in the whole blood of Citrated with 1:2333 diluted esterified tissue factor (Innovin, Siemens Healthcare) (end reaction is made with 1:42000 dilutions) and addition 0.020mL CaCl2To start blood coagulation.All reactions are equal To rerun twice.Monitoring ROTEM parameters simultaneously analyze the ROTEM clotting times (CT).The data that will be collected by device software Microsoft Excel 2010 and/or GraphPad Prism (the 6th edition) are output to for analysis.For every volunteer, According to the undressed sample from identical volunteer, the change hundred in the ROTEM clotting times of the processed sample is calculated Divide ratio.
Experiment (TGA) is generated using fibrin ferment, in the blood plasma rich in hematoblastic blood plasma (PRP) and platelet poor (PPP) dynamics of assessment fibrin ferment generation in, should be rich in the blood plasma (PPP) of hematoblastic blood plasma (PRP) and platelet poor It is according to Hemker, et al., Calibrated automated thrombin generation measurement in clotting plasma.Pathophysiolol Haemost Thromb,33:Blood of the method for 4-15 (2003) from volunteer It is prepared by liquid.Briefly, the whole blood sample that will give the dosage of trial target centrifuges 10 minutes with acquisition in room temperature with 150x g PRP, or 15 minutes are centrifuged to obtain PPP with 2500x g in room temperature.By untapped plasma freezing and it is stored in -80 DEG C. In PRP blood plasma, platelet count is adjusted to every microlitre with autologous PPP and contains 150,000 blood platelet.Immediately in fresh PRP Measure fibrin ferment generation.For PRP samples, 1pM tissue factors (the PRP examinations of 20 μ l are added in the hole of 96 hole microtiter plates Agent, Diagnostica Stago, Inc., Parsippany, NJ) and 80 μ l PRP, repeat three times.By adding 20 μ l's FLUCa buffers (the CaCl of ultimate density 16.7mmol/l2And the Z-Gly-Gly-Arg-AMC fluorometric thrombins of 417mmol/l Substrate) generated to start fibrin ferment.Use Fluoroskan Ascent luminoscope fluorescence intensities.For PPP samples, make With PPP reagents-LOW (ultimate density is the tissue factor and 4 micromolar procoagulant phospholipids of 1pM) replace PRP- reagents and according to The description operation of PRP samples.Using Thrombinoscope software versions 5.0.0.742 (Thrombinoscope BV, Maastricht, The Netherlands) calculate fibrin ferment generation.The data obtained from Thrombinoscope softwares are defeated Go out to Microsoft Excel 2010 and/or GraphPad Prism (the 6th edition) for analysis.In terms of each volunteer, root According to the undressed sample from same volunteer, the percentage of the TGA peak value concentration of thrombin of processed sample is calculated Change.
Factor is diluted on STart4 blood coagulation analyzers (Diagnostica Stago, Parsipanny, NJ) Time (dPT) is analyzed.By the PPP of freezing thaw and give concentration be 1,5,20 and 100nM 106 dosage of anti-TFPI or concentration For the Isotype control antibodies concentration of 100nM.The blood plasma through giving dosage is incubated 30 points at 37 DEG C before dPT measure analyses Clock.50 microlitres of blood plasma is added in 4 cuvettes of STart and is incubated 60 seconds at 37 DEG C.By adding in dilution buffer agent (50mM imidazoles, 0.1M sodium chloride, 1mg/ml bovine serum albumin bletilla 8.34mM CaCl2, pH7.4) in prepare with 1:6000 Diluted tissue factor reagent (Innovin) carrys out activated reactant and in 37 DEG C of preincubate.When 37 DEG C of measurements reach blood coagulation Between, with reaction of reruning twice.Microsoft Excel 2010 or GraphPad Prism (will be output to the clotting times 6 editions) to be analyzed.According to the dPT clotting times of the undressed sample of each volunteer, the sample that product are handled after tested is calculated The change percentage in this dPT clotting times.
As depicted in the figures, as measured by using ROTEM methods, antibody TFPI-106, which causes to derive from, has hemophilia The blood coagulation of the whole blood of the volunteer of A or B is in dose-dependant increase.Specifically, compared with negative control, which shortens blood coagulation Time simultaneously increases maximum clot hardness.In addition, when addition is small rich in blood derived from haemophiliac compared with negative control group In the blood plasma of plate and platelet poor, TFPI-0106 cause fibrin ferment generation in dose-dependant increase, this can by time lag reduce and The peak value concentration of thrombin increase of generation proves.
Figure 10 A and 10B illustrate rush blood coagulations of the TFPI-106 in the whole blood and blood plasma of the volunteer derived from heavy hemophilia A Effect.Figure 10 A show with negative control IgG and be enough to obtain 5%, 10% and 40% normal activity restructuring FVIII it is dense Degree is compared, the influence of the TFPI 106 of two kinds of concentration in ROTEM measure to blood clotting.Figure 10 B are shown and negative control Compared with FVIII, the peak value fibrin ferment generation of the TFPI-106 of three kinds of concentration in rich in hematoblastic blood plasma.The measure is shown Go out the dose dependent clotting time reduction caused by TFPI 106 and peak value fibrin ferment generates increased effect.
Figure 11 A, 11B and 11C illustrate TFPI-106 from resisting with heavy hemophilia A and for the inhibition of FVIII Rush coagulating effectiveness in the whole blood and blood plasma of the volunteer of body (inhibitor).Figure 11 A are shown compared with negative control IgG, two The TFPI-106 of kind concentration is in ROTEM measure to the effect of blood clotting.Figure 11 B are shown compared with negative control IgG, three Peak value fibrin ferment generations of the TFPI-106 of kind concentration in rich in hematoblastic blood plasma.Figure 11 C are shown and negative control IgG Compare, the influence that the TFPI-106 of four kinds of concentration is formed the clot of platelet poor plasma in dilution PT measure.The measure Show that the dose dependent clotting time caused by TFPI 106 is reduced and peak value fibrin ferment generates increased effect.
Figure 12 A, 12B and 12C illustrate TFPI-106 in whole blood and blood plasma from the volunteer with osculant hemophilia A In rush coagulating effectiveness.Figure 12 A show and negative control IgG and are enough the weight for obtaining 5%, 10% and 40% normal activity Group FVIII concentration is compared, the influence of the TFPI 106 of two kinds of concentration in ROTEM measure to blood clotting.Figure 12 B show with Negative control is compared with FVIII, the peak value fibrin ferment generation of the TFPI-106 of three kinds of concentration in rich in hematoblastic blood plasma.Figure 12C shown compared with negative control IgG, and the TFPI-106 of four kinds of concentration is in dilution PT measure to platelet poor plasma shape Into the influence of clot.The measure shows that the dose dependent clotting time caused by TFPI 106 is reduced and peak value fibrin ferment is given birth to Into increased effect.
Figure 13 A, 13B and 13C illustrate TFPI-106 in whole blood and blood plasma from the volunteer with osculant hemophilia B In rush coagulating effectiveness.Figure 13 A show and negative control IgG and are enough the weight for obtaining 5%, 10% and 40% normal activity Group FVIII concentration is compared, and the TFPI 106 of two kinds of concentration is in ROTEM measure to the effect of blood clotting.Figure 13 B show with Negative control is compared with FIX, and the TFPI-106 of three kinds of concentration is to the effect rich in the peak value fibrin ferment generation in hematoblastic blood plasma Fruit.Figure 13 C are shown compared with negative control IgG, and the TFPI-106 of four kinds of concentration is in dilution PT measure to platelet poor Blood plasma forms the influence of clot.The measure shows that the dose dependent clotting time caused by TFPI 106 is reduced and peak value coagulates Hemase generates increased effect.
Figure 14 A, 14B and 14C illustrate TFPI-106 in whole blood and blood plasma from the volunteer with osculant hemophilia A In rush coagulating effectiveness.Figure 14 A show and negative control IgG and are enough the weight for obtaining 5%, 10% and 40% normal activity Group FVIII concentration is compared, and the TFPI 106 of three kinds of concentration is in ROTEM measure to the effect of blood clotting.Each data point generation Table is handled relative to the setting time of the undressed blood sample from identical volunteer with TFPI-106 or FVIII The reduction percentage in the clotting time of the blood sample of single volunteer.Represent the most wide level between the data point of each processing Rod represents the average reduction percentage in the clotting time of volunteer's sample of all tests.Figure 14 B are shown compared with FVIII, The TFPI-106 of three kinds of concentration is to the effect rich in the peak value fibrin ferment generation in hematoblastic blood plasma.Each data point represents phase Generated for the peak value fibrin ferment of the undressed blood sample from identical volunteer, with TFPI-106 or FVIII processing Single volunteer plasma sample peak value fibrin ferment generation increase percentage.Between the data point for representing each processing Most wide horizon bar represents the increase percentage of the peak value fibrin ferment generation of volunteer's sample of all tests.Figure 14 C show four The TFPI-106 of kind concentration is in dilution PT measure to the effect from platelet poor plasma formation clot.Each data point represents Relative to the setting time of the undressed blood sample from identical volunteer, the single volunteer handled with TFPI-106 Plasma sample clotting time reduction percentage.Most wide horizon bar between the data point of each processing represents all tests Volunteer's sample clotting time average reduction percentage.Dilution PT measure shows the agent caused by TFPI 106 The amount dependence clotting time is reduced and the dose dependent peak value shown caused by TFPI 106 is tested in fibrin ferment generation Fibrin ferment generation increase.
Table 33
Sequence
The description of antibody SeqID (recognition sequence) numberings and sequence composition (when referring to specific residue, are numbered using Kabat. VH and VL CDR starting points and end point is defined by using Kabat definition)
Various features of the invention and embodiment mentioned in individual chapters above such as do appropriate variation, according to conjunction Suitable situation is also applied for other chapters and sections.Therefore, the feature illustrated in a chapters and sections can be with illustrating in other chapters and sections Feature combined according to suitable conditions.All reference datas cited herein, including patent, patent application, paper, textbook It is incorporated by reference being incorporated herein with the Sequence accession number enumerated, and references cited therein.When one or more should (include, but are not limited to the term of definition, term is used when the document and similar material that are incorporated to are different or conflicting from the application On the way, described technology, etc.), it is subject to the application.
Sequence table
<110>Pfizer
<120>Tissue factor approach restrainer antibody and application thereof
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Val Pro Ser Leu Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro
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Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn
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Ser Val Ile Gly Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly
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Pro Lys Lys Ile Pro Ser Phe Cys Tyr Ser Pro Lys Asp Glu Gly Leu
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Leu Glu Glu Cys Lys Lys Thr Cys Ile Pro Gly Tyr Glu Lys Thr Ala
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Asn Gln Thr Lys Gln Cys Glu Arg Phe Val Tyr Gly Gly Cys Leu Gly
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Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys Pro Asp Asp
20 25 30
Gly Pro Cys Lys Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr
35 40 45
Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn
50 55 60
Arg Phe Glu Ser Met Glu Glu Cys Lys Lys Val Cys Thr Arg Asp Asn
65 70 75 80
Val Asn Arg Ile Ile Gln Thr Ala Leu Gln Lys Glu Lys Pro Asp Phe
85 90 95
Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg
100 105 110
Tyr Phe Tyr Asn Asn Gln Ser Lys Gln Cys Glu Arg Phe Lys Tyr Gly
115 120 125
Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys
130 135 140
Asn Thr Cys Glu Asp
145
<210> 6
<211> 149
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 6
Ala Ala Glu Glu Asp Glu Glu Phe Thr Asn Ile Thr Asp Ile Lys Pro
1 5 10 15
Pro Leu Gln Lys Pro Thr His Ser Phe Cys Ala Met Lys Val Asp Asp
20 25 30
Gly Pro Cys Arg Ala Tyr Ile Lys Arg Phe Phe Phe Asn Ile Leu Thr
35 40 45
His Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Glu Asn
50 55 60
Arg Phe Glu Ser Leu Glu Glu Cys Lys Glu Lys Cys Ala Arg Asp Tyr
65 70 75 80
Pro Lys Met Thr Thr Lys Leu Thr Phe Gln Lys Gly Lys Pro Asp Phe
85 90 95
Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg
100 105 110
Tyr Phe Tyr Asn Asn Gln Ser Lys Gln Cys Glu Arg Phe Lys Tyr Gly
115 120 125
Gly Cys Leu Gly Asn Leu Asn Asn Phe Glu Ser Leu Glu Glu Cys Lys
130 135 140
Asn Thr Cys Glu Asn
145
<210> 7
<211> 148
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 7
Leu Pro Glu Glu Asp Asp Asp Thr Ile Asn Thr Asp Ser Glu Leu Arg
1 5 10 15
Pro Met Lys Pro Leu His Thr Phe Cys Ala Met Lys Ala Glu Asp Gly
20 25 30
Pro Cys Lys Ala Met Ile Arg Ser Tyr Tyr Phe Asn Met Asn Ser His
35 40 45
Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Arg Gly Asn Lys Asn Arg
50 55 60
Phe Asp Thr Leu Glu Glu Cys Arg Lys Thr Cys Ile Pro Gly Tyr Lys
65 70 75 80
Lys Thr Thr Ile Lys Thr Thr Ser Gly Ala Glu Lys Pro Asp Phe Cys
85 90 95
Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly Phe Met Thr Arg Tyr
100 105 110
Phe Tyr Asn Asn Gln Ser Lys Gln Cys Glu Gln Phe Lys Tyr Gly Gly
115 120 125
Cys Leu Gly Asn Ser Asn Asn Phe Glu Thr Leu Glu Glu Cys Arg Asn
130 135 140
Thr Cys Glu Asp
145
<210> 8
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 8
Gly Gly Gly Ser Gly Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys
1 5 10 15
Ile Glu Trp His Glu Gly Gly Pro Pro His His His His His His His
20 25 30
His His His
35
<210> 9
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 9
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly
20
<210> 10
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 10
Arg Ala Ser Gln Gly Ile Ser Ser Ser Leu Ala
1 5 10
<210> 11
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 11
Ala Ala Ser Thr Leu Gln Ser
1 5
<210> 12
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 12
Gln Gln Leu Asp Ser Tyr Pro Leu Ser
1 5
<210> 13
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 13
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asp Ser Tyr Pro Leu
85 90 95
Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 14
<211> 107
<212> PRT
<213> Homo sapiens
<400> 14
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 15
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 15
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asp Ser Tyr Pro Leu
85 90 95
Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 16
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 16
Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His
1 5 10
<210> 17
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 17
Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln
1 5 10 15
Gly
<210> 18
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 18
Gly Ile Ala Arg Leu Gln Trp Leu Pro Thr Glu Ala Asp Phe Asp Tyr
1 5 10 15
<210> 19
<211> 125
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 19
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ile Ala Arg Leu Gln Trp Leu Pro Thr Glu Ala Asp Phe
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 20
<211> 329
<212> PRT
<213> Homo sapiens
<400> 20
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 21
<211> 454
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 21
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ile Ala Arg Leu Gln Trp Leu Pro Thr Glu Ala Asp Phe
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly
450
<210> 22
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 22
Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His
1 5 10
<210> 23
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 23
Gly Asn Ser Asn Arg Pro Ser
1 5
<210> 24
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 24
Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Val Val
1 5 10
<210> 25
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 25
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Phe Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95
Leu Ser Gly Ser Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu
100 105 110
Gly
<210> 26
<211> 105
<212> PRT
<213> Homo sapiens
<400> 26
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
1 5 10 15
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
20 25 30
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
35 40 45
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
50 55 60
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
65 70 75 80
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
85 90 95
Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 27
<211> 218
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 27
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Phe Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95
Leu Ser Gly Ser Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu
100 105 110
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
115 120 125
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
130 135 140
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
145 150 155 160
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
165 170 175
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
180 185 190
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
195 200 205
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 28
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 28
Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser
1 5 10
<210> 29
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 29
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 30
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 30
Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile
1 5 10
<210> 31
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 31
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 32
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 32
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 33
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 33
Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His
1 5 10
<210> 34
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 34
Gly Asn Ser Asn Arg Pro Ser
1 5
<210> 35
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 35
Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Gly Val
1 5 10
<210> 36
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 36
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95
Leu Ser Gly Ser Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
Gly
<210> 37
<211> 218
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 37
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95
Leu Ser Gly Ser Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
115 120 125
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
130 135 140
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
145 150 155 160
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
165 170 175
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
180 185 190
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
195 200 205
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 38
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 38
Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser
1 5 10
<210> 39
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 39
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 40
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 40
Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile
1 5 10
<210> 41
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 41
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 42
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 42
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 43
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 43
Ser Gly Ser Thr Ser Asn Ile Gly Thr Met Tyr Val His
1 5 10
<210> 44
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 44
Arg Asn Asn His Arg Pro Ser
1 5
<210> 45
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 45
Leu Ala Trp Asp Asp Thr Leu Arg Ala Tyr Val
1 5 10
<210> 46
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 46
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly
100 105 110
<210> 47
<211> 216
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 47
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 48
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 48
Gly Leu Thr Ile Asp Asn Tyr Ala Met Gln
1 5 10
<210> 49
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 49
Gly Ile Ser Gly Asn Ser Arg Ser Ile Gly Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 50
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 50
Phe Leu His Glu Ser Asp Tyr
1 5
<210> 51
<211> 116
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 51
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Thr Ile Asp Asn Tyr
20 25 30
Ala Met Gln Trp Val Arg Gln Arg Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asn Ser Arg Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Ile Asp Ser Leu Arg Ala Asp Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ile Phe Leu His Glu Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 52
<211> 445
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 52
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Thr Ile Asp Asn Tyr
20 25 30
Ala Met Gln Trp Val Arg Gln Arg Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asn Ser Arg Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Ile Asp Ser Leu Arg Ala Asp Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ile Phe Leu His Glu Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 53
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 53
Thr Gly Ser Ser Ser Asn Leu Gly Ala Asp Tyr Asp Val Gln
1 5 10
<210> 54
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 54
Gly Asn Asn Asn Arg Pro Ser
1 5
<210> 55
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 55
Gln Ser Tyr Asp Arg Ser Leu Ser Gly Ser Met Val
1 5 10
<210> 56
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 56
Gln Ser Val Leu Thr Gln Pro Pro Ser Leu Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Leu Gly Ala Asp
20 25 30
Tyr Asp Val Gln Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Phe Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asn Tyr Tyr Cys Gln Ser Tyr Asp Arg Ser
85 90 95
Leu Ser Gly Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
Gly
<210> 57
<211> 218
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 57
Gln Ser Val Leu Thr Gln Pro Pro Ser Leu Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Leu Gly Ala Asp
20 25 30
Tyr Asp Val Gln Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Phe Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asn Tyr Tyr Cys Gln Ser Tyr Asp Arg Ser
85 90 95
Leu Ser Gly Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
115 120 125
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
130 135 140
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
145 150 155 160
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
165 170 175
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
180 185 190
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
195 200 205
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 58
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 58
Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser
1 5 10
<210> 59
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 59
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 60
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 60
Asn Gly Ala Ala Ala Ala Trp Asp Tyr
1 5
<210> 61
<211> 118
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 61
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Asn Gly Ala Ala Ala Ala Trp Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 62
<211> 447
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 62
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Asn Gly Ala Ala Ala Ala Trp Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 63
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 63
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 64
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 64
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 65
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 65
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 66
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 66
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Leu Gly Ala Thr Ser Leu Ser Ala Phe Asp Ile Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly
<210> 67
<211> 116
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 67
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Thr Ile Asp Asn Tyr
20 25 30
Ala Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asn Ser Arg Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ile Phe Leu His Glu Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 68
<211> 445
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 68
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Thr Ile Asp Asn Tyr
20 25 30
Ala Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asn Ser Arg Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ile Phe Leu His Glu Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 69
<211> 116
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 69
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Ile Asp Asn Tyr
20 25 30
Ala Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asn Ser Arg Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ile Phe Leu His Glu Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 70
<211> 445
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 70
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Ile Asp Asn Tyr
20 25 30
Ala Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asn Ser Arg Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ile Phe Leu His Glu Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 71
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 71
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly
100 105 110
<210> 72
<211> 216
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 72
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 73
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 73
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly
100 105 110
<210> 74
<211> 216
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 74
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 75
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 75
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly
100 105 110
<210> 76
<211> 216
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 76
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 77
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 77
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly
100 105 110
<210> 78
<211> 216
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 78
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Thr Met
20 25 30
Tyr Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Asn Asn His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Ala Trp Asp Asp Thr Leu
85 90 95
Arg Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu
115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
130 135 140
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
145 150 155 160
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205
Thr Val Ala Pro Thr Glu Cys Ser
210 215
<210> 79
<211> 116
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 79
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Thr Ile Asp Asn Tyr
20 25 30
Ala Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asn Ser Arg Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ile Phe Leu His Glu Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 80
<211> 445
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 80
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Thr Ile Asp Asn Tyr
20 25 30
Ala Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Ser Gly Asn Ser Arg Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ile Phe Leu His Glu Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe
225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 81
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 81
Lys Ala Ser Gln Asp Val His Thr Ala Val Ala
1 5 10
<210> 82
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 82
Trp Ala Ser Thr Arg His Thr
1 5
<210> 83
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 83
Gln Gln His Tyr Thr Thr Pro Tyr Thr
1 5
<210> 84
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 84
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Cys Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys
100 105
<210> 85
<211> 106
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 85
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
1 5 10 15
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
20 25 30
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
35 40 45
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
65 70 75 80
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
85 90 95
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 86
<211> 213
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 86
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Cys Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Ala Asp Ala Ala Pro
100 105 110
Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly
115 120 125
Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn
130 135 140
Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn
145 150 155 160
Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
165 170 175
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr
180 185 190
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe
195 200 205
Asn Arg Asn Glu Cys
210
<210> 87
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 87
Gly Tyr Thr Phe Thr Asp Tyr Asn Leu Asp
1 5 10
<210> 88
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 88
Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 89
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 89
Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr
1 5 10
<210> 90
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 90
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Ile Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ala Ser Val Thr Val Ser Ser
115
<210> 91
<211> 324
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 91
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
1 5 10 15
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
115 120 125
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
145 150 155 160
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
165 170 175
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
210 215 220
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
<210> 92
<211> 443
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 92
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Ile Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ala Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr
115 120 125
Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu
130 135 140
Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp
145 150 155 160
Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser
180 185 190
Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser
195 200 205
Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys
210 215 220
Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr
245 250 255
Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser
260 265 270
Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg
275 280 285
Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile
290 295 300
Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn
305 310 315 320
Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
325 330 335
Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu
340 345 350
Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe
355 360 365
Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala
370 375 380
Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr
385 390 395 400
Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly
405 410 415
Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His
420 425 430
Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440
<210> 93
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 93
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Cys Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 94
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 94
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Ile Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Ala Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 95
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 95
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 96
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 96
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 97
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 97
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Ile Ser Val Asp Gln Ala Lys Asn Ser Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 98
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 98
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Ile Ser Val Asp Gln Ala Lys Asn Ser Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 99
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 99
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 100
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 100
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 101
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 101
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 102
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 102
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 103
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 103
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Gln Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 104
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 104
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Gln Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 105
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 105
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 106
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 106
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 107
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 107
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Ile Ser Val Asp Gln Ala Lys Asn Ser Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Ile Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 108
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 108
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Leu Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Asp Ile Asn Pro Ile Asn Gly Ala Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Ile Ser Val Asp Gln Ala Lys Asn Ser Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Ile Tyr Tyr Gly Asp Tyr Asp Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 109
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 109
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 110
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 110
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 111
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 111
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 112
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 112
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 113
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 113
Lys Ala Ser Gln Asp Val Ile Thr Ala Val Ala
1 5 10
<210> 114
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 114
Trp Ala Ser Thr Arg His Thr
1 5
<210> 115
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 115
Gln Gln His Tyr Ser Thr Pro Tyr Thr
1 5
<210> 116
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 116
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Val Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ile Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 117
<211> 213
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 117
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Val Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ile Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ala Asp Ala Ala Pro
100 105 110
Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly
115 120 125
Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn
130 135 140
Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn
145 150 155 160
Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
165 170 175
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr
180 185 190
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe
195 200 205
Asn Arg Asn Glu Cys
210
<210> 118
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 118
Gly Tyr Thr Phe Thr Asp Tyr Thr Met Asp
1 5 10
<210> 119
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 119
Asp Ile Asn Pro Ser Asn Gly Gly Ser Ile Tyr Asn Arg Lys Phe Lys
1 5 10 15
Gly
<210> 120
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 120
Met His Tyr Asn Tyr Asp Gly Phe Pro Tyr
1 5 10
<210> 121
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 121
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Ser Asn Gly Gly Ser Ile Tyr Asn Arg Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Met His Tyr Asn Tyr Asp Gly Phe Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 122
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 122
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Ser Asn Gly Gly Ser Ile Tyr Asn Arg Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Met His Tyr Asn Tyr Asp Gly Phe Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 123
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 123
Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Thr Tyr Met His
1 5 10 15
<210> 124
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 124
Leu Ala Ser Asn Leu Glu Ser
1 5
<210> 125
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 125
Gln His Ile Arg Glu Leu Pro Phe Thr
1 5
<210> 126
<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 126
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Ala Tyr Tyr Cys Gln His Ile Arg
85 90 95
Glu Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 127
<211> 217
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 127
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Ala Tyr Tyr Cys Gln His Ile Arg
85 90 95
Glu Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Ala
100 105 110
Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
115 120 125
Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro
130 135 140
Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn
145 150 155 160
Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr
165 170 175
Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His
180 185 190
Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile
195 200 205
Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215
<210> 128
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 128
Gly Tyr Thr Phe Thr Ser Tyr Val Met His
1 5 10
<210> 129
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 129
Tyr Leu Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 130
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 130
Thr Leu Leu Tyr Ala Met Asp Tyr
1 5
<210> 131
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 131
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Leu Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Ser Leu Ile Ser Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Thr Leu Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly Ser Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 132
<211> 358
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 132
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Leu Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Ser Leu Ile Ser Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Thr Leu Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly Ser Ser
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met
355
<210> 133
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 133
Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala His
1 5 10
<210> 134
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 134
Tyr Asp Asn Asn Arg Pro Ser
1 5
<210> 135
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 135
Gln Ser Trp Asp Asp Gly Val Pro Val
1 5
<210> 136
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 136
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Tyr
35 40 45
Tyr Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Asp Gly Val Pro Val
85 90 95
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 137
<211> 212
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 137
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Tyr
35 40 45
Tyr Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Asp Gly Val Pro Val
85 90 95
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala
100 105 110
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
115 120 125
Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val
130 135 140
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu
145 150 155 160
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser
165 170 175
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser
180 185 190
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro
195 200 205
Thr Glu Cys Ser
210
<210> 138
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 138
Gly Phe Thr Phe Arg Ser Tyr Gly Met Ser
1 5 10
<210> 139
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 139
Ser Ile Arg Gly Ser Ser Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 140
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 140
Lys Tyr Arg Tyr Trp Phe Asp Tyr
1 5
<210> 141
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 141
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Arg Gly Ser Ser Ser Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Lys Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 142
<211> 446
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 142
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Arg Gly Ser Ser Ser Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Lys Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 143
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 143
Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala His
1 5 10
<210> 144
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 144
Tyr Asp Val Asn Arg Pro Ser
1 5
<210> 145
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 145
Gln Ser Trp Trp Asp Gly Val Pro Val
1 5
<210> 146
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 146
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe
35 40 45
Tyr Asp Val Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Trp Asp Gly Val Pro Val
85 90 95
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 147
<211> 212
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 147
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe
35 40 45
Tyr Asp Val Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Trp Asp Gly Val Pro Val
85 90 95
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala
100 105 110
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
115 120 125
Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val
130 135 140
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu
145 150 155 160
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser
165 170 175
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser
180 185 190
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro
195 200 205
Thr Glu Cys Ser
210
<210> 148
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 148
Gly Phe Thr Phe Arg Ser Tyr Gly Met Asp
1 5 10
<210> 149
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 149
Ser Ile Arg Gly Ser Arg Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 150
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 150
Leu Tyr Arg Tyr Trp Phe Asp Tyr
1 5
<210> 151
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 151
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30
Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Arg Gly Ser Arg Ser Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 152
<211> 446
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 152
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30
Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Arg Gly Ser Arg Ser Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
130 135 140
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
210 215 220
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val
225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
<210> 153
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 153
Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala His
1 5 10
<210> 154
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 154
Tyr Asp Asn Asn Arg Pro Ser
1 5
<210> 155
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 155
Gln Ser Trp Asp Asp Gly Val Pro Val
1 5
<210> 156
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 156
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Tyr
35 40 45
Tyr Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Asp Gly Val Pro Val
85 90 95
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 157
<211> 105
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 157
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
1 5 10 15
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
20 25 30
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
35 40 45
Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
50 55 60
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
65 70 75 80
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
85 90 95
Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 158
<211> 212
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 158
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Tyr
35 40 45
Tyr Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Asp Asp Gly Val Pro Val
85 90 95
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala
100 105 110
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
115 120 125
Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val
130 135 140
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu
145 150 155 160
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser
165 170 175
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser
180 185 190
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro
195 200 205
Thr Glu Cys Ser
210
<210> 159
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 159
Gly Phe Thr Phe Ser Asn Tyr Ala Leu Ser
1 5 10
<210> 160
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 160
Ser Ile Ser Ser Gly Gly Ala Thr Tyr Tyr Pro Asp Ser Val Glu Gly
1 5 10 15
<210> 161
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 161
Gly Ala Tyr Gly Ser Asp Tyr Phe Asp Tyr
1 5 10
<210> 162
<211> 118
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 162
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Leu Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Ser Gly Gly Ala Thr Tyr Tyr Pro Asp Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Gln Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr
85 90 95
Arg Gly Ala Tyr Gly Ser Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 163
<211> 441
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 163
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Leu Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Ser Gly Gly Ala Thr Tyr Tyr Pro Asp Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Arg Asn Ile Leu Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Gln Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr
85 90 95
Arg Gly Ala Tyr Gly Ser Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
115 120 125
Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr
180 185 190
Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser
195 200 205
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro
210 215 220
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
225 230 235 240
Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys
245 250 255
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
260 265 270
Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu
275 280 285
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met
290 295 300
His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser
305 310 315 320
Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335
Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln
340 345 350
Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe
355 360 365
Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu
370 375 380
Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
385 390 395 400
Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
405 410 415
Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr
420 425 430
Glu Lys Ser Leu Ser His Ser Pro Gly
435 440
<210> 164
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 164
Lys Ser Ser Gln Ser Leu Leu Glu Ser Asp Gly Lys Thr Tyr Leu Asn
1 5 10 15
<210> 165
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 165
Leu Val Ser Ile Leu Asp Ser
1 5
<210> 166
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 166
Leu Gln Ala Thr His Phe Pro Gln Thr
1 5
<210> 167
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 167
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Glu Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Val Ser Ile Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Ala
85 90 95
Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 168
<211> 219
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 168
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Glu Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Leu Val Ser Ile Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Ala
85 90 95
Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 169
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 169
Gly Phe Thr Phe Ser Asn Tyr Ala Met Ser
1 5 10
<210> 170
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 170
Thr Ile Ser Arg Ser Gly Ser Tyr Ser Tyr Phe Pro Asp Ser Val Gln
1 5 10 15
Gly
<210> 171
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 171
Leu Gly Gly Tyr Asp Glu Gly Asp Ala Met Asp Ser
1 5 10
<210> 172
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 172
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Arg Ser Gly Ser Tyr Ser Tyr Phe Pro Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Gly Tyr Asp Glu Gly Asp Ala Met Asp Ser Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 173
<211> 327
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 173
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 174
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 174
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Arg Ser Gly Ser Tyr Ser Tyr Phe Pro Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu Gly Gly Tyr Asp Glu Gly Asp Ala Met Asp Ser Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445
<210> 175
<211> 360
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 175
gaggtgcagc tgctggagtc tggcggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gattctggga 300
gctacttcgt tatcggcttt tgatatctgg ggccaaggga caatggtcac cgtctcgagc 360
<210> 176
<211> 336
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 176
cagtctgtgc tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccatc 60
tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120
cttccaggaa cagcccccaa actcctcatc tatggtaaca gcaatcggcc ctcaggggtc 180
cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc 240
caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gagtggttca 300
ggggtattcg gcggagggac caagctgacc gtccta 336
<210> 177
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 177
gaggtgcagc tgctggagtc tggcggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gattctggga 300
gctacttcgt tatcggcttt tgatatctgg ggccaaggga caatggtcac cgtctcgagc 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgctggggca 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc cccgggt 1347
<210> 178
<211> 654
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 178
cagtctgtgc tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccatc 60
tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120
cttccaggaa cagcccccaa actcctcatc tatggtaaca gcaatcggcc ctcaggggtc 180
cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc 240
caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct gagtggttca 300
ggggtattcg gcggagggac caagctgacc gtcctaggtc agcccaaggc tgccccctcg 360
gtcactctgt tcccgccctc ctctgaggag cttcaagcca acaaggccac actggtgtgt 420
ctcataagtg acttctaccc gggagccgtg acagtggcct ggaaggcaga tagcagcccc 480
gtcaaggcgg gagtggagac caccacaccc tccaaacaaa gcaacaacaa gtacgcggcc 540
agcagctatc tgagcctgac gcctgagcag tggaagtccc acagaagcta cagctgccag 600
gtcacgcatg aagggagcac cgtggagaag acagtggccc ctacagaatg ttca 654

Claims (31)

1. the epitope of the Kunitz domains 2 (K2) of one kind specific binding tissue factor approach restrainer (TFPI) is separated Antibody or its antigen-binding fragment, are selected from the residue of the following group wherein the epitope includes：
(a) according to SEQ ID NO：Residue Ile105, Arg107 and Leu131 of 2 numberings；
(b) according to SEQ ID NO：2 numbering residue Ile105, Arg107, Leu131 and one or more be selected from the residual of the following group Base：Cys106, Gly108, Cys130 and Gly132；
(c) according to SEQ ID NO：2 numbering residue Ile105, Arg107, Leu131 and one or more be selected from the residual of the following group Base：Asp102, Arg112, Tyr127, Gly129, Met134 and Glu 138；
(d) according to SEQ ID NO：2 numbering residue Ile105, Arg107, Leu131 and one or more be selected from the residual of the following group Base：Cys106, Gly 108, Cys130 and Gly132, and further include one or more and be selected from the residue of the following group：Asp102、 Arg112, Tyr127, Gly129, Met134 and Glu138.
2. the antibody of claim 1 or its antigen-binding fragment, it includes following heavy (H) chain numbered according to Kabat and gently (L) Chain residue：H33Ala、H58Tyr、H95Leu、H96Gly、H97Ala、H98Thr、H99Ser、H100Leu、H100A Ser、 L29Ala, L31Tyr, L91Tyr, L95A Ser and L95B Gly.
3. the antibody of claim 2 or its antigen-binding fragment, it includes the following residue numbered according to Kabat：
(a) H33 is Ala or Val；
(b) H47 is Trp；
(c) H50 is Ala；
(d) H51 is Ile；
(e) H52 is Ser, Arg, Lys, Phe or Tyr；
(f) H56 is Ser, Arg or Lys；
(g) H58 is Tyr；
(h) H95 is Leu；
(i) H96 is Gly, Ala, Arg, Asn, Lys, Pro, Ser or Val；
(j) H97 is Ala；
(k) H98 is Thr, His, Ile, Leu, Met, Phe or Tyr；
(1) H99 is Ser；
(m) H100 is Leu, Phe, Trp or Tyr；
(n) H100A is Ser, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, Phe, Pro or Trp；
(o) L29 is Ala；
(p) L31 is Tyr；
(q) L91 is Tyr；
(r) L95A is Ser, Phe, Trp or Tyr；
(s) L95B is Gly；And
(t) L95C is Ser, Arg, Asn, Gln, Glu, Ile, Leu, Lys, Met, Phe, Trp, Tyr or Val；
And optionally include following residue：(u) L93 is Ser；
(v) L96 is Gly.
4. a kind of separated antibody or its antigen-binding fragment of the K2 of specific binding TFPI, wherein the antibody is selected from by wrapping The group formed containing following antibody：
(a) VH of heavy chain variable region (VH) complementary determining region 1 (CDR-H1), CDR-H2 and CDR-H3, the CDR-H1 bags are included The NO of ID containing SEQ：38 amino acid sequence, the CDR-H2 include SEQ ID NO：39 amino acid sequence and the CDR-H3 Include SEQ ID NO：40 amino acid sequence；
(b) SEQ ID NO are included：The VH of 41 amino acid sequence；
(c) SEQ ID NO are included：The VH of 63 amino acid sequence；
(d) VL of light chain variable region (VL) complementary determining region 1 (CDR-L1), CDR-L2 and CDR-L3, the CDR-L1 bags are included The NO of ID containing SEQ：33 amino acid sequence, the CDR-L2 include SEQ ID NO：34 amino acid sequence and the CDR-L3 Include SEQ ID NO：35 amino acid sequence；
(e) SEQ ID NO are included：The VL of 36 amino acid sequence；
(f) SEQ ID NO are included：The heavy chain constant region (CH) of 20 amino acid sequence；
(g) SEQ ID NO are included：The constant region of light chain (CL) of 26 amino acid sequence；
(h) SEQ ID NO are included：The heavy chain of 64 amino acid sequence；
(i) SEQ ID NO are included：The heavy chain of 42 amino acid sequence；
(j) SEQ ID NO are included：The light chain of 37 amino acid sequence；
(k) SEQ ID NO are included：The CDR-H1 of 48 amino acid sequence, include SEQ ID NO：49 amino acid sequence CDR-H2 and include SEQ ID NO：The CDR-H3 of 50 amino acid sequence；
(l) VL of CDR-L1, CDR-L2 and CDR-L3 are included, the CDR-L1 includes SEQ ID NO：43 amino acid sequence, The CDR-L2 includes SEQ ID NO：The 44 amino acid sequence and CDR-L3 includes SEQ ID NO：45 amino acid sequence Row；
(m) VH comprising CDR-H1, CDR-H2 and CDR-H3 and VL, the CDR- comprising CDR-L1, CDR-L2 and CDR-L3 H1 includes SEQ ID NO：38 amino acid sequence, the CDR-H2 include SEQ ID NO：39 amino acid sequence and described CDR-H3 includes SEQ ID NO：40 amino acid sequence, and the CDR-L1 include SEQ ID NO：33 amino acid sequence Row, the CDR-L2 include SEQ ID NO：The 34 amino acid sequence and CDR-L3 includes SEQ ID NO：35 amino acid Sequence；
(n) SEQ ID NO are included：The VH of 63 amino acid sequence and include SEQ ID NO：The VL of 36 amino acid sequence；And
(o) by SEQ ID NO：Heavy chain that 64 amino acid sequence is formed and by SEQ ID NO：37 amino acid sequence institute group Into light chain.
5. the antibody of claim 1 or its antigen-binding fragment, wherein the epitope is further included according to SEQ ID NO：2 One or more of numbering are selected from the residue of the following group：Glu100, Glu101, Asp102, Gly104 and Tyr109.
6. a kind of separated antibody or its antigen-binding fragment of the K2 of specific binding TFPI, wherein the epitope includes basis SEQ ID NO：Residue Glu101, Pro103 of 2 numbering, Tyr109, Thr111, Ser119, Gln121, Glu123, Arg124, Lys126 and Leu 140.
7. the antibody of claim 6 or its antigen-binding fragment, it includes：
(a) VH of CDR-H1, CDR-H2 and CDR-H3 are included, the CDR-H1 includes SEQ ID NO：87 amino acid sequence, The CDR-H2 includes SEQ ID NO：The 88 amino acid sequence and CDR-H3 includes SEQ ID NO：89 amino acid sequence Row；And
(b) VL of CDR-L1, CDR-L2 and CDR-L3 are included, the CDR-L1 includes SEQ ID NO：81 amino acid sequence, The CDR-L2 includes SEQ ID NO：The 82 amino acid sequence and CDR-L3 includes SEQ ID NO：83 amino acid sequence Row.
8. the antibody of claim 1 or its antigen-binding fragment, wherein the antibody is selected from the following group：
(a) antibody according to the Kabat at least one paratope residues numbered is included, the paratope residue is located at It is following according to SEQ ID NO：At least one epitope residues on the TFPI of 2 numberingsIt is interior：Epitope residues 102Asp is positioned at anti- Former paratope residue H58Tyr'sIt is interior；Epitope residues 104Gly is located at paratope residue H58Tyr'sIt is interior； Epitope residues 105Ile be located at paratope residue H33Ala, H50Ala, H51Ile, H52Ser, H56Ser, H58Tyr and H95Leu'sIt is interior；Epitope residues 106Cys is located at paratope residue H100Leu and H100A Ser'sIt is interior； Epitope residues 107Arg is located at paratope residue H96Gly, H97Ala, H98Thr, H99Ser and H100Leu's It is interior；Epitope residues 108Gly is located at paratope residue H100Leu'sIt is interior；It is mutual that epitope residues 112Arg is located at antigen Cover residue L29Ala's and L31TyrIt is interior；Epitope residues 127Tyr is located at paratope residue L31Tyr's It is interior；Epitope residues 129Gly is located at paratope residue L31Tyr'sIt is interior；Epitope residues 130Cys is located at antigen complementation Position residue L91Tyr and L95B Gly'sIt is interior；Epitope residues 131Leu be located at paratope residue H47Trp, H50Ala, H58Tyr, L95A Ser, L95B Gly and L95C SerIt is interior；Epitope residues 132Gly is located at antigen complementation Position residue H58Tyr and L95A Ser'sIt is interior；Epitope residues 134Met is located at paratope residue L95A Ser'sIt is interior；And epitope residues 138Glu is located at paratope residue L29Ala'sIt is interior；
(b) comprising at least one paratope residue numbered according to Kabat, the paratope residue can be with following According to SEQ ID NO：The epitope residues of the TFIP of 2 numberings form hydrogen bond：Epitope residues 102Asp can be with paratope residue H58Tyr forms hydrogen bond；Epitope residues 107Arg can form hydrogen bond with least one be selected from the paratope residue of the following group： H96Gly, H97Ala, H98Thr, H99Ser and H100Leu；Epitope residues 112Arg can be with paratope residue L29Ala shapes Into hydrogen bond；Epitope residues 127Tyr can form hydrogen bond with paratope residue L31Tyr；And epitope residues 131Leu can be with resisting Former paratope residue L95B Gly form hydrogen bond；And
(c) antibody according to the Kabat at least one paratope residues numbered is included, the paratope residue includes It is following due to according to SEQ ID NO：The interaction of the epitope residues of 2 numberings and non-zero in the surface region hidden becomes Change：Epitope residues 102Asp and paratope residue H58Tyr interacts；Epitope residues 104Gly and paratope are residual Base H58Tyr interacts；Epitope residues 105Ile is interacted with least one be selected from the paratope residue of the following group： H33Ala, H34Met, H50Ala, H51Ile, H52Ser, H56Ser, H58Tyr and H95Leu；Epitope residues 106Cys with least One is selected from the paratope residue interaction with the following group：H95Leu, H100Leu, H100A Ser and L91Tyr；Epitope Residue 107Arg is interacted with least one be selected from the paratope residue of the following group：H96Gly、H97Ala、H98Thr、 H99Ser and H100Leu；Epitope residues 108Gly and paratope residue H100Leu interacts；Epitope residues 112Arg Interacted with least one be selected from the paratope residue of the following group：L29Ala, L31Tyr and L93Ser；Epitope residues 127Tyr is interacted with least one be selected from the paratope residue of the following group：L31Tyr and L95B Gly；Epitope residues 129Gly is interacted with least one be selected from the paratope residue of the following group：H100A Ser, L31Tyr and L91Tyr； Epitope residues 130Cys is interacted with least one be selected from the paratope residue of the following group：H95Leu、H100A Ser、 L31Tyr, L91Tyr and L95B Gly；Epitope residues 131Leu is selected from the paratope residue phase of the following group with least one Interaction：H47Trp, H50Ala, H58Tyr, H95Leu, L31Tyr, L91Tyr, L95A Ser, L95B Gly, L95C Ser and L96Gly；Epitope residues 132Gly is interacted with least one be selected from the paratope residue of the following group：H58Tyr and L95A Ser；Epitope residues 133Asn and paratope residue L95A Ser interact；Epitope residues 134Met with least One is selected from the paratope residue interaction with the following group：L93Ser, L94Ser and L95A Ser；And epitope residues 138Glu is interacted with least one be selected from the paratope residue of the following group：L28Gly, L29Ala and L93Ser.
9. a kind of separated monoclonal antibody, its competition binding TFPI and/or with the antibody of claim 4 or its antigen binding fragment Section combines identical epitope.
10. the antibody of claim 1 or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment are with about 1 × 10- 8M to about 1 × 10-10Binding affinity (Kd) the value combination TFPI of M.
11. the antibody of claim 1 (a) or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment：(i) subtract Few clotting time as measured by the dilution prothrombin time based on blood plasma；(ii) reduce and such as surveyed by Thrombectomy The whole blood coagulation time of amount；(iii) increase fibrin ferment to produce；(iv) FXa activity of the increase in the presence of TFPI；(V) strengthens Platelet accumulation in the presence of TFPI；(vi) fibrin of the increase in the presence of TFPI produces；Or (vii) its is any Combination.
12. the antibody of claim 11 or its antigen-binding fragment, wherein the blood plasma or whole blood lack Factor IX or the factor IX。
13. a kind of separated nucleic acid molecules, it includes the antibody of coding claim 1 or the nucleotides sequence of its antigen-binding fragment Row.
14. a kind of separated nucleic acid molecules, it includes the nucleotide sequence of the antibody of coding claim 4.
15. the nucleic acid molecules of claim 14, wherein the nucleotide sequence coded VH and VL, the VH include CDR-H1, CDR-H2 and CDR-H3 and the VL include CDR-L1, CDR-L2 and CDR-L3, and the CDR-H1 includes SEQ ID NO：38 Amino acid sequence, the CDR-H2 include SEQ ID NO：The 39 amino acid sequence and CDR-H3 includes SEQ ID NO：40 Amino acid sequence, and the CDR-L1 includes SEQ ID NO：33 amino acid sequence, the CDR-L2 include SEQ ID NO：The 34 amino acid sequence and CDR-L3 includes SEQ ID NO：35 amino acid sequence.
16. the nucleic acid molecules of claim 15, nucleotide sequence coded SEQ ID NO are included wherein described：63 amino acid sequence The VH of row and include SEQ ID NO：The VL of 36 amino acid sequence.
17. the nucleic acid molecules of claim 14, it includes selected from the nucleotide sequence of the following group：
(a)SEQ ID NO：175 sequence；
(b)SEQ ID NO：176 sequence；
(c)SEQ ID NO：177 sequence；
(d)SEQ ID NO：178 sequence；
(e) it is present in the Insert Fragment sequence of the plasmid of ATCC numbering PTA-122328 preservations；And
(f) it is present in the Insert Fragment sequence of the plasmid of ATCC numbering PTA-122329 preservations.
18. a kind of pharmaceutical composition, it includes the antibody of claim 1 or its antigen-binding fragment and pharmaceutically acceptable biography Pass body or excipient.
19. one kind reduces the active method of tissue factor approach restrainer (TFPI), it includes applied to object in need The antibody of the claim 1 of therapeutically effective amount or its antigen-binding fragment.
20. one kind reduces the active method of tissue factor approach restrainer (TFPI), it includes applied to object in need The claim 4 (m) or the antibody of 4 (n) or its antigen-binding fragment of therapeutically effective amount.
21. the method for claim 19, the method includes the antibody using claim 7.
22. a kind of method for shortening the bleeding time, it includes the claim 1 that therapeutically effective amount is applied to object in need Antibody or its antigen-binding fragment.
23. a kind of method for shortening the bleeding time, it includes the claim 4 that therapeutically effective amount is applied to object in need (m) or the antibody of 4 (n) or its antigen-binding fragment.
24. the method for claim 22, the method includes the antibody using claim 7.
25. the method for claim 22, wherein the object suffers from or easily with hemophilia A, hemophilia B, vascular blood friend Sick (vWD) or blood platelet disorders.
26. the method for claim 23, wherein the object suffers from or easily with hemophilia A, hemophilia B, vascular blood friend Sick (vWD) or blood platelet disorders.
27. the method for claim 19, further includes the FVIIa using therapeutically effective amount.
28. the method for claim 20, further includes the FVIIa using therapeutically effective amount.
29. a kind of method for shortening the bleeding time in object in need, the method include the power using therapeutically effective amount Profit requires 1 antibody or its antigen-binding fragment and coagulant.
30. the method for claim 29, wherein the object suffers from or easily suffer from hemophilia A or hemophilia B, and the blood coagulation Agent is selected from following：Factor VIIa, Factor IX, factors IX and tranexamic acid.
31. the method for claim 30, the method includes the antibody or antigen binding fragment using claim 4 (m) or 4 (n) Section.
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